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International Immunology Advance Access originally published online on July 14, 2006
International Immunology 2006 18(9):1355-1362; doi:10.1093/intimm/dxl068
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Modulation of matrix metalloproteinase-9 (MMP-9) secretion in B lymphopoiesis

Doron Melamed1,2,3, Orit Messika1,3,4, Lea Glass-Marmor4 and Ariel Miller1,2,4

1 Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
2 Rappaport Family Institute for Research in the Medical Sciences, Haifa, Israel
3 Department of Immunology, Technion, Haifa, Israel
4 Neuroimmunology Unit, Carmel Medical Center, 7 Michal Street, Haifa 34362, Israel

Correspondence to: A. Miller; E-mail: millera{at}tx.technion.ac.il


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix, thus involved in cellular migration. The extent and role of MMPs secretion in primary non-transformed B cells, and specifically during early stages of development in the bone marrow (BM), has been barely unveiled. Herein, we investigated the secretion of MMP-9 during B lymphopoiesis and its modulation in response to different mitogens and cytokines. To do so, we used our BM culture system and well-studied mutated mouse models to isolate the different B cell populations. Our results show that MMP-9 is spontaneously secreted throughout B lymphopoiesis, and that the level of secreted MMP-9 is developmentally regulated. Using reverse transcription–PCR, we found that IFNßR is expressed throughout B cell development, while tumor necrosis factor (TNF)-{alpha}R-p55 and IFN{gamma}R expressions are initiated only at the pre-B stage. We found that TNF{alpha} stimulates MMP-9 secretion in transitional cells, whereas IFNs suppress MMP-9 secretion in immature cells. LPS and phorbol 12-myristate 13-acetate suppressed MMP-9 secretion in transitional cells, whereas LPS and concanavalin A stimulated MMP-9 secretion in mature B cells. We conclude that B lymphocyte development is accompanied with MMP-9 secretion and the developing cells are competent to modify this secretion upon different immune stimuli.

Keywords: autoimmune, B cells, cytokines, gelatinase, hematopoiesis, inflammation, migration


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The development of B lymphocytes in the bone marrow (BM) is limited by positive and negative selection events aiming to test the competence of the antigen receptor (1). Developmental progression of cells that fail to express a receptor or expressing signaling incompetent receptors or self-reactive receptors is aborted and such cells undergo receptor editing or elimination by apoptosis (1, 2). In contrast, a functional non-self receptor promotes positive selection and migration from the BM to the periphery (3, 4). Such cells enter the follicular niches in the spleen for final differentiation and selection into the long-lived pool (5). It is not completely understood how this migration is regulated. Data suggest an important role for chemokines such as stromal cell-derived factor-1{alpha} and B lymphocyte chemoattractant in B cell generation in the BM and migration to the peripheral lymphoid tissue (6, 7), as well as responsiveness to survival factors such as B cell-activating factor (BAFF) (2). In addition to these factors, appropriate migration of B lymphocytes also depends on their ability to leave the circulation and to penetrate into tissues. A major limit for this extravasation is the basement membrane of the capillaries, which requires proteolytic cleavage of components such as collagens (CL) (predominantly type IV CL) and glycoproteins such as laminin (8).

During inflammatory process, mature activated B cells migrate to the injured tissue and site of inflammation (9). This process is mediated by the high endothelial venules, which support extensive lymphocyte extravasation from the blood (10), and the ability of the B cells to degrade the basement membrane and the extracellular matrix (ECM) (11). Thus, in B lymphocytes, both normal homeostasis and inflammatory responses depend on migration processes, which require secretion of proteolytic enzymes capable to degrade ECM.

Matrix metalloproteinases (MMPs) play a critical role in the degradation of the ECM and in facilitating T cell migration into the target tissues (12, 13). The MMPs constitute a family of zinc-dependent endopeptidases whose members have been implicated in many physiological and pathological processes (14). Among the different subclasses of MMPs, are the gelatinases A (MMP-2) and B (MMP-9), which degrade various types of denatured ECM proteins (13, 15). MMP-2 and MMP-9 are produced by many white blood cells with cell-specific pattern (16, 17). We and others have shown that MMP's expression is modulated by soluble mediators such as growth factors and cytokines (12, 13, 18, 19), and their activation is regulated by proteolysis while inactivation is regulated by the endogenous tissue inhibitors of MMPs (14, 15). Pro-MMP9 has been shown to bind beta 2 integrin of leukocytes (20), suggesting that cell surface-tethered MMP-9 may also function in directing migration by mediating cleavage of receptors and/or chemokines. In addition to MMP secretion, T lymphocyte migration is facilitated by amoeboid shape change and contact guidance (21). However, both surface-tethered MMP-9 and the protease-independent mechanism of migration have never been shown to function in B lymphocytes.

Despite the importance of migration ability to B cell homeostasis and B cell responses, there are scarce reports of studies probing MMPs synthesis and function in the B lineage. Earlier studies have utilized transformed B cell line to suggest that MMP-9 protein is the primary family member that is constitutively produced by peripheral human B cells (22, 23). Activation of these transformed B cells with mitogens or with pro-inflammatory cytokines such as IL-1, IL-8 and tumor necrosis factor {alpha} (TNF{alpha}) stimulates MMP-9 secretion (22, 23). In contrast, anti-inflammatory cytokines, such as TGFß (22), or IFNs (type I or type II) (24) suppress MMP-9 secretion by these transformed B cells. Despite of these results, the extent of MMP-9 secretion in normal B cells remains unclear. In addition, it is unknown whether MMP-9 is also secreted at earlier stages of B lymphopoiesis. This is particularly important as recent studies suggest that earlier precursor B cells are recruited from the BM to the spleen upon immunization (25–27) and at early stages of B cell leukemia (28, 29). Herein, we used our BM culture system and well-defined mouse models to study MMP-9 secretion in developing B cells. We found a constitutive secretion of MMP-9 in developing B cells, which was inversely correlated with the stage of development and modulated by stimuli provided by mitogens and cytokines.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
Normal B10.D2nSn/J, 3-83Tg B10.D2nSn/J (3-83Tg), 3-83TgCD19–/– B10.D2nSn/J (3) and C57/BL6-µMT/µMT (µMT) mice (30) were used for these experiments. Mice were housed and bred at the animal facility of the Technion, Faculty of Medicine (Haifa, Israel), and used at 4–10 weeks of age. The use of animals for these studies has been reviewed and approved by the Technion Committee for Care and Use of Laboratory Animals.

Cell culture and B cell purification
Mature B cells were purified from the spleen of normal mice (B10.D2) after negative selection by magnetic beads (B cell isolation kit, Miltenyi Biotec). Early stages of B cell development were prepared using our BM culture system (31). This culture system allows preferential growth of B cell precursors from pro-B to the transitional stage. All cells in the culture are AA4.1 positive, and mature B cells, AA4.1-/IgMlo/IgDhi are not developing in these cultures. Briefly, BM cells, RBC depleted, were cultured for 5 days in the presence of 50–100 U ml–1 rIL-7 in IMDM with 10% FCS. Transitional B cells were purified from BM culture of 3-83Tg mice. Immature B cells were purified from BM culture of 3-83TgCD19–/– mice. Pre-B cells were purified from BM culture of normal mice after negative selection of Ig-expressing cells using MACS magnetic beads (Miltenyi Biotec) and polyclonal goat anti-mouse {kappa} and goat anti-mouse {lambda} antibodies (Southern Biotechnology Associates, Brimingham, AL, USA). Pro-B cells were purified from BM cultures of µMT mice. For zymography and ELISA measurements, 5 x 105 cells were cultured for 24 h in a serum-free medium (Bio-Target, Biological Industries, Beit H'aemek, Israel) containing 0.2% BSA, 100 U ml–1 penicillin, 10 µg ml–1 streptomycin, 4 mM L-glutamine, 1% sodium pyruvate and 1% non-essential amino acid. After culture, cell viability was determined by both trypan blue exclusion method and tetrazolium hydroxide (XTT) viability assay [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] according to the manufacturer's instructions (Biological Industries). Protein secretion was adjusted to cell viability.

Reagents
Murine rTNF-{alpha}, murine rIFN-{gamma} and murine rIFN-ß were purchased from R&D Systems, USA. Phorbol 12-myristate 13-acetate (PMA), LPS and concanavalin A (ConA) were purchased from Sigma, USA. Cells were cultured in 96-well microtiter plates, for 24 h, in serum-free medium with or without stimulation.

Detection of MMP-9 in supernatants
Detection of MMP-9 in supernatants was carried out using a gelatinolytic activity assay as we have previously described (18, 19). Briefly, supernatants (24 µl volume per sample) collected from cells stimulated or unstimulated (control) were analyzed by zymography, which enables to detect zymogenic and activated forms of the gelatinases MMP-9 and MMP-2. Secreted proteins were separated by electrophoresis in SDS-PAGE containing 0.1% gelatin. After electrophoresis, gels were denatured by incubation in 2.5% Triton X-100 for 30 min, incubated overnight in substrate buffer (50 mmol Tris–HCl, pH 7.5, containing 10 mmol CaCl2) at 37°C and stained with 0.5% Commassie brilliant blue. Molecular weight markers (Bio-Rad Laboratories) and positive control of r-MMP-2 and MMP-9 (Chemicon) were run with each gel. Computerized densitometry was used to evaluate relative enzymatic activity (Nonlinear Dynamics Limited, UK, Renium; TotalLab software). For some experiments, determination of pro-MMP-9 protein was carried out using commercial ELISA (R&D systems), according to the manufacturer's instructions. Plates were read in an ELISA reader at 450 nm and pro-MMP-9 protein concentration was determined using a reference standard curve.

RNA purification and cDNA analysis
Total RNA was purified from cultured cells or spleen cells as we have described (3). RNA samples were reverse transcribed to cDNA and PCR amplified to detect TNF{alpha}R-1 (p55), IFN{gamma}R-1 and IFNßR-1. PCR conditions for all samples were: 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, for 32 cycles. Primer sequences for TNF{alpha}R-1 (p55) were—sense: 5'-GACT GATTCCTGCACCAACATT-3' and anti-sense: 5'-ATCTGCTGCACCAAGTGCC-3'; for IFN{gamma}R-1 were—sense: 5'-GACTGATTCCTGCACCAACATT-3' and anti-sense: 5'-TTTACCACAGAGAGCAAGGACT-3'; and for IFNßR-1 were—sense: 5'-GACCTCAACTACATGGTCTACA-3' and anti-sense: 5'-ACT CCACGACATACTCAGCAC-3'. The PCR products were fractionated on 1% agarose.

Flow cytometry
Single-cell suspensions from spleen or BM cultures were stained for surface marker expression using FITC-, PE- and biotin-conjugated antibodies, visualized with streptavidin Tri Color (Caltag Laboratories). Antibodies used for cell staining were: B220, clone RA3-6B2; anti-CD43, clone S7 (Southern Biotechnology Associates, Inc.); anti-IgDa, clone AMS 9.1; goat-anti-mouse IgM (Caltag, CA, USA); goat anti-mouse {kappa} and goat anti-mouse {lambda} antibodies (Southern Biotechnology Associates, Inc.). Data for three-color analysis were collected on a FACSCaliburTM and analyzed using CELLQuestTM software (Becton Dickinson). For all analysis, forward and side scatter gates were set to include viable cells and exclude dead cells and debris.

Statistical evaluation
The statistical significance of differences between experimental groups was determined using one sample two-tailed signed-rank's test (Wilcoxon), with differences considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Constitutive secretion of MMP-9 in developing B cells is inversely correlated with the stage of development
MMP-9 is produced by mature B cells and is important for their physiological function (22, 23). We investigated if MMP-9 is also produced at earlier stages of B cell lymphopoiesis, and whether or not B cells at this stage of development are competent to secrete MMP-9 upon stimulation. In order to do such analysis, purified B cell populations from each stage of development have been prepared. We thought that cell sorting using antibodies to specific surface markers should not be applicable as antibody–ligand interactions can stimulate MMP-9 secretion. To overcome this limitation, we used our BM culture system and different, but well-described, mutated mouse models to prepare pro-B, pre-B, immature, transitional and mature B cells. The pro-B cell population was grown in BM culture of Ig-µ-deficient mice (µMT). These mice carry a stop codon mutation in the Igµ transmembrane tail exon and, as a consequence, B cell development in these mice is blocked at the pro-B stage (30). As shown in Fig. 1 (top, left panel), cells grown in µMT BM culture express the B220+/CD43+ pro-B phenotype. The pre-B cell population was prepared from BM culture of normal mice. After removing the IgM+ cells, the resulting IgM– population contains predominantly pre-B cells as confirmed by their surface marker phenotype B220+/CD43– (Fig. 1, top, right panel). To prepare immature and transitional cells, we used our Ig transgenic mouse model (3-83Tg). Due to accelerated development imposed by the rearranged transgenic receptor, most of the B cells grown in the 3-83Tg BM culture are late immature or transitional, expressing the IgMhi/IgDlo phenotype (Fig. 1, bottom, middle panel) (31), and are AA4.1+ (data not shown). However, as we have previously shown, in the absence of CD19, 3-83Tg B cell development is blocked at the immature stage (3). Hence, cells grown in BM culture of 3-83Tg/CD19–/– mice are considered immature as the majority of the cells are IgMhi/IgD– (Fig. 1, bottom, left panel). Mature B cells, which are not developing in these BM cultures, were purified untouched from spleens of normal mice and are expressing the phenotype of IgMlo/IgDhi (Fig. 1, bottom, right panel). Mature B cells were purified from spleen of normal mice.


Figure 1
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  		Fig. 1 Phenotype of developing B cell populations. (A) An illustrated phenotype of developing B lymphocytes. (B) Typical surface marker expression of the different B cell populations that were prepared. Pro-B cells were purified from BM cultures of µMT mice and are characterized by co-expression of B220 and CD43. Pre-B cells were purified from BM culture of a normal mouse after negative selection of IgM-expressing cells by magnetic beads. Pre-B cells are characterized as IgM–/B220+/CD43–. Immature B cells were purified from BM culture of 3-83TgCD19–/– mice. Immature B cells are characterized as IgM+/IgD–. Transitional B cells were purified from BM culture of 3-83Tg mice. Transitional B cells are characterized as IgM+/IgD+. Mature B cells were purified from the spleen of normal mice after negative selection by magnetic beads. Mature B cells are characterized as IgM+/IgD+. Preparation of BM culture and cell purification is described in Methods.

 
To test for spontaneous MMP-9 secretion, B cells from the different stages of development were incubated for 24 h in serum-free medium. Supernatants were collected and analyzed for MMP-9 by zymography. As shown in Fig. 2(A), gelatinolytic activity at ~97 kDa corresponding to the molecular mass of pro-MMP-9 was detected in conditioned medium from all B cell populations. To quantify the level of MMP-9, densitometric analysis was performed. The results in Fig. 2 show relative levels of MMP-9 in developing B cells compared with that produced by mature splenic B cells. We found that levels of spontaneous MMP-9 secretion are inversely correlated with developmental progression. The zymography assay (Fig. 2A and B) revealed that at early stages of B cell development, prior to B cell receptor (BCR) expression (pre-B and pro-B), a significant 2- to 4-fold more MMP-9 was spontaneously secreted. In contrast, spontaneous MMP-9 secretion at the later stages of development (immature and transitional B cells) was very low and not significantly different from that secreted by mature B cells. The use of 10 mM EDTA completely inhibited MMP-9 zymography activity, implicating the specificity and competence of this assay (data not shown). Nevertheless, these results were independently confirmed using MMP-9-specific ELISA (Fig. 2C). It should be noted that cell death rates after 24 h of incubation was relatively low (20–25%) with no significant differences between different B cell populations


Figure 2
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  		Fig. 2 MMP-9 is spontaneously secreted throughout B lymphopoiesis. The indicated B cell populations were prepared as described in Fig. 1 legend. Cells were cultured for 24 h in serum-free medium and supernatants were collected to determine MMP-9. (A) Gelatin zymography of pro-MMP-9 from culture. The gel shown is representative of three different experiments. (B) Zymography gels were subjected to densitometric analysis to obtain quantitative measurement of pro-MMP-9 activity in the supernatants. The results from three different experiments are presented as mean ± SD. (C) Supernatants were independently assayed for quantitative pro-MMP-9 by specific ELISA. The results from three different experiments are presented as mean ± SD.

 
Mitogen-mediated MMP-9 modulation in B lymphopoiesis
Since MMP-9 secretion is induced by mitogens, cytokines and phorbol ester (12, 13), we tested whether developing B lymphocytes can be induced to secrete MMP-9 in response to these stimuli. To do so, B lymphocytes from the different developmental stages were incubated for 24 h in serum-free medium and stimulated with LPS (1–100 µg ml–1), PMA (1–100 ng ml–1) or ConA (0.1–2 µg ml–1). Cell viability monitored by trypan blue exclusion method and by the XTT assay revealed survival rate of >90% (data not shown), and MMP-9 secretion in supernatants was adjusted to it. The results in Fig. 3 show that stimulation of pro-B, pre-B and immature B cells with LPS, PMA or ConA (at all concentration tested) did not significantly changed the basal level of secreted MMP-9. However, stimulation of transitional B cells with LPS (50 µg ml–1) or PMA (100 ng ml–1) resulted in significant 50 and 40% reduction, respectively, in MMP-9 secretion, and no effect had been observed upon stimulation with ConA (since concentration >0.5 µg ml–1 resulted in low cell viability after 24 h). Significant increase of 50 and 220% in MMP-9 secretion was detected in mature B cells stimulated with LPS (100 µg ml–1) or ConA (2 µg ml–1), respectively, while no effect was detected in mature B cells stimulated with PMA.


Figure 3
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  		Fig. 3 MMP-9 secretion in response to mitogenic stimulation. The indicated B cell populations were prepared as described in Fig. 1 legend. Cells were cultured for 24 h in serum-free medium in the absence or presence of LPS (100 µg ml–1 for the mature cells and 50 µg ml–1 for the other cell types), PMA (10 ng ml–1 for the mature cells and 100 ng ml–1 for the other cell types) and ConA (2 µg ml–1 for the mature cells and 0.5 µg ml–1 for the other cell types), and supernatants were collected to determine MMP-9 by zymography. Gels were subjected to densitometric analysis to obtain quantitative measurement of pro-MMP-9 activity in the supernatants. Shown are the results for individual experiments and as group means, for each B cell population. An asterisk marks significant differences with P < 0.05.

 
Expression of cytokine receptors in developing B cells
To test whether developing B cells can secrete MMP-9 in response to different cytokine stimulation, we have first determined cytokine receptor expression in these cells. To do so, mRNA samples from each cell population were tested by reverse transcription–PCR for the detection of TNF{alpha}R-1 (p55), IFN{gamma}R-1 and IFNßR-1. As shown in Fig. 4, IFNßR-1 was detected throughout B cell development (pro-B, pre-B, immature, transitional and mature B cells). Expression of TNF{alpha}R-1 and IFN{gamma}R-1 initiated at the pre-B stage and both receptors were found in immature transitional and mature B cells.


Figure 4
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  		Fig. 4 Expression of cytokine receptor in B lymphopoiesis. The different B cell populations were purified as described in Fig. 1 legend and used to prepare cDNA. The expression of TNF{alpha}R, IFN{gamma}R and IFNßR in each B cell population was determined by reverse transcription–PCR using specific primers. The gel shown is a representative of three different experiments.

 
Cytokine-mediated MMP-9 modulation in B cells
To test for cytokine-mediated MMP-9 responsiveness, B cell populations were incubated for 24 h in serum-free medium containing of TNF{alpha} (1–100 ng ml–1), IFN{gamma} (100–2000 U ml–1) or IFNß (100–2000 U ml–1). Cell viability revealed a survival rate of >90% (data not shown), and MMP-9 secretion in supernatants was adjusted to it. The effect of cytokines on MMP-9 secretion is summarized in Fig. 5. Stimulation of pro-B, pre-B and mature B cells with TNF{alpha}, IFN{gamma} or IFNß did not change significantly the basal secretion of MMP-9 at any tested concentration. Stimulation of immature B cells with IFNß (1000 U ml–1) or with IFN{gamma} (1000 U ml–1) resulted in a significant 50 and 35% reduction (respectively) in MMP-9 secretion (and lesser in concentrations of 500 U ml–1 or 2000 U ml–1), whereas no effect was found upon stimulation with TNF{alpha} (at any concentration). In contrast, TNF{alpha} (10 ng ml–1) induced a significant 36% increase of MMP-9 secretion in transitional B cells, while no significant effect was detected when these cells were treated with either IFN{gamma} or IFNß (at any concentration).


Figure 5
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  		Fig. 5 Secretion of MMP-9 in response to different cytokine stimulation. The indicated B cell populations were prepared as described in Fig. 1 legend. Cells were cultured for 24 h in serum-free medium in the absence or presence of TNF{alpha} (100 ng ml–1 for the mature cells and 10 ng ml–1 for the other cell types), IFN{gamma} (1000 U ml–1) or IFNß (1000 U ml–1), and supernatants were collected to determine MMP-9 by zymography. Gels were subjected to densitometric analysis to obtain quantitative measurement of pro-MMP-9 activity in the supernatants. Shown are the results for individual experiments and as group means, for each B cell population. An asterisk marks significant differences with P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Earlier studies have shown that MMP-9 is important for homeostasis and functional responses of immune cells. In the B lineage, however, most of these studies have focused on mature cells or utilized transformed B cell lines. In the current study, we bring evidence from experiments with non-transformed B cells showing that MMP-9 is produced at early stages of B cell development, and that its production is modified in response to cytokines and mitogens. Thus, MMP-9 may have an important function for maintaining normal B lymphopoiesis. Importantly, due to experimental limitations, the different B cell sub-populations studied herein, representing the various stages of B cell development, have been prepared from different mutated mouse models. Although these mouse models have been very well studied and characterized, further studies of normal B lymphopoiesis, in mice and humans, should be carried out to exclude a potential effect of the mutation on this process.

In addition to Ig gene rearrangement, B lymphopoiesis is regulated by intercellular contact with stromal cells and the stromal cell-derived cytokine IL-7 (32–34). The availability of these factors is facilitated by the establishment of specific cellular niches and movement of the developing B cells between these niches (35). MMP-9 mediates release of sKit-Ligand, which helps to recruit stem and progenitor cells, and activation of MMP-9 serves as a decisive checkpoint for the entry of stem cells into the S phase of cell cycle (36). Interestingly, earlier stages of B cell development predominantly depend on the niche microenvironment (33, 35). In accordance with this, we show here that early-developing B cells constitutively synthesize high level of MMP-9, which may facilitate their entry and residing in the niche. Later stages of B cell development are less dependent on the niche microenvironment, which is also correlated with a drop in the levels of spontaneously secreted MMP-9. This developmental regulation of MMP-9 synthesis may be interrelated with its physiological function. It is possible that secretion of MMP-9 is important for the recruitment and proliferation of early progenitors into the B-lymphopietic niches as a homeostatic drive to maintain production of B lymphocytes. Thus, MMP-9 may contribute to cell-cycle regulation and maturation of B lymphocytes. This notion is supported by the fact that both B cell proliferation (33) and MMP-9 secretion (Fig. 2) decline with developmental progression. We interpret these results to suggest that in normal developing B cells, high levels of MMP-9 are correlated with cell proliferation, whereas the low levels produced later in development, maintain homeostasis and survival. This function of MMP-9 can yet be compensated by other mechanisms, as MMP-9-deficient mice are viable (37). However, upon acute BM ablation, hematopoietic recovery is severely impaired in MMP-9–/– mice (36). Yet, studies of B cell development and their immune responsiveness to antigenic stimulation in MMP9–/– mice have not been reported.

Responsiveness to cytokines and mitogens have been fairly well studied in mature B cells, and are less known in developing B cells. Studies have shown that immature B cells respond to LPS by antibody production is not different from mature B cells (38). Despite this, we found that only transitional and mature B cells increase MMP-9 secretion in response to LPS PMA or ConA. This may reflect a protective mechanism to minimize polyclonal activation and responsiveness in order to prevent autoimmunity. Studies have shown that >60% of the immature B cells express a self-reactive receptor (39), and polyclonal stimulation of these cells in response bacterial antigens may lead to the synthesis of auto-antibodies. Furthermore, we and others have recently shown that developing B cells are competent to undergo class switch recombination (40) and somatic hyper mutation (41), suggesting that B cells acquire the ability to mount an immune responsiveness early in development. Hence, controlling the immune responsiveness of this B cell compartment is essential to prevent autoimmunity. One mechanism that may contribute in controlling the potential autoimmunity harm is the lack of MMP-9 responsiveness in early stages of B cell development following stimulation with LPS.

The expression of cytokine receptor mRNA explains the responsiveness of developing B cells to cytokine stimulation. For example, IFNs suppress B lymphopoiesis (42, 43), TNF{alpha} imposes growth arrest and apoptosis of early and immature precursors (44), whereas BAFF is a survival and maturation factor (2). It is not clear how these cytokines exerts their effect on developing B cells. One potential mechanism is by modifying secretion of factors such as MMP-9, which may change the microenvironment milieu, as by degrading the ECM and release of growth factors embedded in it (45). The fact that only the late stages of B cell development modify MMP-9 secretion in response to TNF{alpha}, IFNß or IFN{gamma} suggests that such responsiveness is developmentally regulated and acquired with maturation. Similarly, quantitative and qualitative differences in BCR signaling have been described during B lymphopoiesis, thereby leading to different outcome upon receptor ligation (46). Interestingly, mature B cells fail to secrete MMP-9 in response to any of the cytokine stimulation we used here. This is in contrast to their known responsiveness to pro and anti-inflammatory cytokines during inflammation and/or autoimmune responses (47). It is possible that MMP-9 secretion in mature B cells is regulated by activation. Thus, resting splenic B cells fail to secrete MMP-9 in response to cytokine stimulation (our results), whereas activated B cells in vivo are highly responsive to these stimuli [reviewed in (47)].

The data shown here were obtained using well-defined transgenic and knockout mouse models to allow collection of specific B cell subsets untouched. As these are abnormal conditions for B cell development, selection and function, it will be important to confirm these results in a more physiological system.

MMP-9 is stimulated by inflammation and has an important pathological role in many clinical disorders such as cancer and autoimmunity. In several B cell malignancies, MMP-9 levels are correlated with the disease prognosis. Studies have shown that high levels of MMP-9 are detected in serum of B-cell chronic lymphocytic leukemia patients (24, 48) and in high grade of non-Hodgkin lymphoma (49). In these malignancies, the high level of MMP-9 was found to correlate with the poor prognosis (49). On the other hand, poor prognosis of pro/pre-B cell malignancies such as B-cell acute lymphoblastic leukemia is correlated with low MMP-9 production (28, 29). MMP-9 is implicated also in autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. The patients affected by these immune (T and B cell)-mediated disorders produce high levels of MMP-9 in the periphery and at the target organ (50–52). Levels of MMP-9 secretion increase during the relapsing of the autoimmune disease and down-regulate by immunotherapies (53). As other members of the MMps' family, MMP-9 may exert some of its effects in these inflammatory processes by modulation of immune mediators such as cytokines, chemokines and cell-surface receptor expression. Examples include: processing of IL-8 while degradation of certain CXC chemokines (13), shedding of membrane TNF{alpha} or CD95 (54). This is in addition to its ability to degrade the ECM and to release growth factors embedded in it (45). Similarly, MMP-9 may be involved in modulating responsiveness of B lymphocytes to several immune stimuli including those mediated by cytokines and chemokines. Thus, the secretion of MMPs by B cells may have an important role in feedback loops and immunoregulatory networks for B cells lymphopoiesis activation and chemotaxis.


   Abbreviations
 
BAFF, B cell-activating factor
BCR, B cell receptor
BM, bone marrow
CL, collagen
ConA, concanavalin A
ECM, extracellular matrix
MMP, matrix metalloproteinase
PMA, phorbol 12-myristate 13-acetate
TNF, tumor necrosis factor

   Notes
 
Transmitting editor: D. Wallach

Received 2 March 2006, accepted 16 June 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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