Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2011 Jul 10.
Published in final edited form as: Arthritis Rheum. 2007 Jun;56(6):1776–1786. doi: 10.1002/art.22697

Tumor Necrosis Factor α Activates Release of B Lymphocyte Stimulator by Neutrophils Infiltrating the Rheumatoid Joint

Lakhvir K Assi 1, See Heng Wong 1, Andreas Ludwig 2, Karim Raza 1, Caroline Gordon 1, Michael Salmon 1, Janet M Lord 1, Dagmar Scheel-Toellner 1
PMCID: PMC3132453  EMSID: UKMS35847  PMID: 17530706

Abstract

Objective

The tumor necrosis factor (TNF) family member B lymphocyte stimulator (BLyS) is an important regulator of B cell–dependent autoimmunity. Similar to other TNF family members, it is generally expressed as a transmembrane protein and cleaved from the surface to release its active soluble form. This study was undertaken to investigate the expression of BLyS and regulation of BLyS release from the surface of neutrophils infiltrating the rheumatoid joint.

Methods

BLyS expression was studied in neutrophils from the synovial fluid and peripheral blood of patients with rheumatoid arthritis (RA) and healthy controls, by flow cytometry, Western blotting, and immunofluorescence analyses. Peripheral blood neutrophils cultured with 50% RA synovial fluid were study for membrane expression of BLyS. Neutrophils were exposed to a range of proinflammatory cytokines to study the mechanisms of surface loss of BLyS.

Results

Expression of BLyS was detected on the surface of peripheral blood neutrophils from both RA patients and healthy controls, whereas BLyS expression on synovial fluid neutrophils was very low. Constitutive expression of BLyS was observed in neutrophils, both on the cell membrane and in intracellular stores; however, BLyS release from each of these sites was found to be regulated independently. Of the various cytokine stimuli, only TNFα triggered release of BLyS from the neutrophil membrane. This process led to release of physiologically relevant quantities of soluble BLyS, which was dependent on the presence of the pro-protein convertase furin. In contrast, stimulation of neutrophils with granulocyte colony-stimulating factor induced BLyS release from the intracellular stores. Incubation of peripheral blood neutrophils with RA synovial fluid led to TNFα-dependent shedding of BLyS from the cell surface.

Conclusion

These findings indicate that as neutrophils enter the site of inflammation, they release surface-expressed BLyS in a TNFα-dependent manner, and thus may contribute to local stimulation of autoimmune B cell responses.

Neutrophils are abundant, short-lived effectors of the innate immune system. During a disease flare in a rheumatoid joint, large numbers of neutrophils invade the synovial fluid. Although their role is not completely understood, the release of proteases and reactive oxygen species by neutrophils is likely to contribute to joint damage (1). Neutrophils are known to release proinflammatory factors, including leukotriene B4 (LTB4), interleukin-8 (IL-8), and IL-1β. Among these, LTB4 production by neutrophils is a prerequisite for the development of arthritis in the K/BxN serum transfer model of arthritis (2,3). In addition, the development of collagen-induced arthritis in DBA/1 mice depends on the presence of neutrophils in the rheumatoid joint (3,4). However, it is widely held that rheumatoid arthritis (RA) is not caused by the dysregulation of a single cell population, but is a multifactorial disease involving inappropriate actions and interactions between cells of the innate and the adaptive immune systems.

In this context, a recent study provided evidence that neutrophils can produce B lymphocyte stimulator (BLyS; trademark of Human Genome Sciences, Rockville, MD) (5), a B cell–stimulating cytokine. There is mounting evidence for a role of B cells in the complex interactions leading to the development of RA. While it has been known for some time that production of rheumatoid factor is closely associated with RA, it is now recognized that the combined presence of rheumatoid factor and anti–cyclic citrullinated peptide antibodies has a very high specificity for the development of chronic RA in patients with very early synovitis (6,7). These findings suggest a key role for B cells in all stages of RA and indicate that factors involved in the activation or survival of autoimmune B cells may be of crucial importance for disease progression.

BLyS (also known as BAFF, TALL-1, THANK, or zTNF4 [8–12]) is a member of the tumor necrosis factor (TNF) superfamily that regulates several aspects of B cell physiology, including proliferation, survival, and differentiation. It is therefore not surprising that BLyS has been proposed as an important regulator of B cell–dependent autoimmune diseases. The observation that BLyS-transgenic mice develop autoimmune disorders, and the fact that levels of BLyS in RA and other autoimmune disorders are elevated indicate that the expression of BLyS must be tightly regulated to maintain B cell survival without triggering autoimmunity (13–15).

When production of BLyS by dendritic cells was first described, it was suggested that BLyS, similar to other members of the TNFα gene family, is expressed as a type II single transmembrane protein that forms biologically active trimers (9,10,12). To release the cytokine, it is cleaved from the cell surface. The main proteases responsible for the release of TNFα are TACE and ADAM-17, although other proteases, such as proteinase 3, can also mediate TNFα release (16). However, the release of BLyS from the cell surface appears to be regulated in a different manner.

The multibasic motif of the stalk region of BLyS (R-N-K-R) resembles the target sequence for furin, a member of the pro-protein convertase family, prompting the notion that BLyS release is mediated by furin (17). Interestingly, while BLyS is produced as a membrane-bound pro-form in most myeloid cell types, recent studies have shown that it can also be expressed in a readily processed form in intracellular vesicles in granulocyte colony-stimulating factor (G-CSF)–primed neutrophils (5). The regulation of these distinct stores of BLyS in neutrophils and their potential role in autoimmune diseases such as RA or lupus have not been described.

In the present study, we investigated the expression of BLyS by neutrophils from the synovial fluid and the peripheral blood of patients with RA. Initial experiments showed a significantly lower expression of BLyS on the surface of synovial fluid neutrophils compared with peripheral blood neutrophils. Consequently, we investigated the mechanisms involved in the loss of BLyS from the surface of neutrophils. We exposed neutrophils to a range of proinflammatory cytokines. Intriguingly, we observed rapid release of surface-bound BLyS in cells exposed to TNFα and slow release of BLyS from intracellular sources upon long-term exposure to G-CSF. These observations suggest that neutrophils have 2 distinct BLyS stores, an intracellular store that is sensitive to G-CSF, and a membrane-bound store that is released in the presence of TNFα. Exposure of peripheral blood neutrophils to synovial fluid from RA patients resulted in a rapid, TNFα-dependent release of BLyS from the neutrophil surface. We conclude that there is a TNFα-dependent mechanism leading to the rapid release of BLyS from the neutrophil surface during infiltration of neutrophils into the rheumatoid joint. This release may well contribute to the B cell autoimmunity observed in RA.

PATIENTS AND METHODS

Patients

All study patients (n = 11) fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 revised criteria for RA (18). The median disease duration was 5 years (range 10 weeks to 31 years). Six patients were taking the following disease-modifying antirheumatic drugs (DMARDs): hydroxychloroquine (n = 1), sulfasalazine (n = 1), methotrexate (n = 2), methotrexate plus sulfasalazine (n = 1), and gold (n = 1). Five patients were not taking DMARDs at the time of clinical presentation and entry into the study. The median age of the RA patients was 57 years (range 32–88 years). Nine of the 11 patients were female, and 10 were rheumatoid factor positive. All patients and healthy volunteer control subjects (n = 9) gave their written informed consent for participation in this study, which was approved by the local research ethics committee.

Purification of neutrophils

Highly purified neutrophils were isolated by Percoll gradient separation as described previously (19). The neutrophils were resuspended in RPMI 1640 medium (Gibco BRL, Grand Island, NY) containing 10% fetal calf serum (Sera Lab International, Crowley Down, UK), penicillin (100 units/ml), streptomycin (100 μg/ml), and 2 mM l-glutamine (Sigma-Aldrich, Poole, UK) to a final cell density of 2 × 106/ml. The purity of the neutrophil preparations was checked by light microscopy, after staining neutrophil cytospins with a commercial May-Grünwald Giemsa stain kit (Diff-Quik; Baxter Healthcare, Compton, UK). Purity of the neutrophil preparations was routinely >97%, with the major contaminant being eosinophils. Neutrophils from synovial fluid were isolated with a similar protocol, after pretreatment of the synovial fluid with 10 units/ml hyaluronidase for 20 minutes at 37°C to reduce viscosity.

Purification of monocytes

Monocytes were isolated from the peripheral blood of normal healthy donors by magnetic-activated cell sorting using superparamagnetic microbeads (MACS beads) labeled with anti-CD14 antibody (Miltenyi, Bergisch Gladbach, Germany). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of normal healthy donors, incubated with anti-CD14–labeled beads, washed, and separated from the unlabeled cells in a magnetized column (Miltenyi) according to the manufacturer’s instructions.

Cytokine stimulation of neutrophils

Neutrophils, at a density of 2 × 106/ml, were treated with a range of cytokines (BioSource Invitrogen, Carlsbad, CA) in varying concentrations as follows: G-CSF at 1,000 units/ml, granulocyte–macrophage CSF (GM-CSF) at 100 ng/ml, TNFα at 10 ng/ml, IL-6 at 250 ng/ml, IL-1β at 50 ng/ml, interferon-β at 1,000 units/ml, and IL-8 at 200 ng/ml. For studies of the mechanism of surface release of BLyS, neutrophils were treated with TNFα (10 ng/ml). The cells were cultured at 37°C in a humidified incubator with an atmosphere of 5% CO2. In selected experiments, neutrophils were precultured for 30 minutes in the presence or absence of a range of protease inhibitors, including the furin convertase inhibitor chloromethylketone at 25 μM (Calbiochem, Nottingham, UK), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) at 2 mM (Sigma-Aldrich), the metalloprotease inhibitor GW280264X at 10 μM (GlaxoSmithKline, Stevenage, UK), elafin at 70 nM, and both an elastase inhibitor and cathepsin G inhibitor, each at 200 nM (Calbiochem) (20).

Measurement of BLyS and TNFα by enzyme-linked immunosorbent assay (ELISA)

Expression of BLyS and TNFα by neutrophils was determined using specific ELISAs. The commercial BLyS-specific ELISA (R&D Systems, Abingdon, UK) and TNFα-specific ELISA (Becton Dickinson, Franklin Lakes, NJ) were carried out according to the manufacturer’s instructions.

Flow cytometric analysis of BLyS, furin, and CD95 expression

Extracellular BLyS expression was analyzed on the surface of the neutrophils isolated from RA patients or control individuals. Neutrophils were resuspended with anti-BLyS monoclonal antibody (anti-BAFF; PeproTech, London, UK) or mouse IgG negative control (Dako, Ely, UK) for 1 hour on ice. All antibodies were prediluted in phosphate buffered saline (PBS) containing 2% bovine serum albumin (BSA). All washing steps were performed in PBS containing 2% BSA. The primary antibody was detected with fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG1 secondary antibody (Southern Biotechnology, Birmingham, AL). The same staining conditions were applied to monocytes, Epstein-Barr virus (EBV)–transformed B cell lines, and cells of the promyelocytic cell line HL-60. Specificity of the anti-BLyS monoclonal antibody was validated by preincubating the antibody for 1 hour with recombinant human BLyS at a concentration of 10 μg/ml.

Furin expression was determined by staining of control and TNFα-treated neutrophils with a goat anti-human furin antibody (R&D Systems) and detection with an FITC-labeled rabbit anti-goat secondary antibody (Southern Biotechnology). CD95 expression was detected using a murine monoclonal antibody (clone CH11; Upstate Bioscience, Charlottesville, VA) and detection with an FITC-conjugated goat anti-mouse IgM antibody (Southern Biotechnology). All experiments were controlled using species-, isotype-, and concentration-matched irrelevant antibodies. Samples were read using a Coulter Epics XL Flow Cytometer (Beckman Coulter, Fullerton, CA). Samples were subsequently analyzed using WinMDI software (Scripps Research Institute, San Diego, CA), with results expressed as the mean fluorescence intensity.

Exposure of blood neutrophils to RA synovial fluid

Neutrophils from healthy volunteers were cultured in the presence of 50% RA synovial fluid and 50% RPMI 1640 medium for 1 hour at 37°C, with or without neutralizing antibodies against TNFα (BioSource Invitrogen). As controls, neutrophils were stimulated with TNFα in the presence of anti-TNFα antibodies. The cultured cells were then assessed for membrane expression of BLyS in the same manner as described above.

Immunofluorescence analysis of intra- and extracellular expression of BLyS by neutrophils

Extracellular and intracellular BLyS expression was analyzed by immunofluorescence confocal analysis. To specifically detect the membrane-bound pool of BLyS, live neutrophils were stained with an anti-BLyS monoclonal antibody (PeproTech) or isotype control antibody (Dako), which was used at the same protein concentration as the anti-BLyS antibody, for 1 hour. This was followed by 2 wash steps using PBS for 5 minutes. Specific staining was detected using an FITC-labeled anti-rabbit secondary antibody (Southern Biotechnology). The cells were then fixed and permeabilized using the Caltag Fix and Perm Kit (BioSource Invitrogen) according to the manufacturer’s instructions.

The permeabilized cells were stained with a mouse anti-BLyS monoclonal antibody and revealed with Texas Red–conjugated goat anti-mouse IgG1. The stained cells were cytocentrifuged onto glass slides, using a Shandon II cytocentrifuge (Pittsburgh, PA), and allowed to air dry for a minimum of 3 hours. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole, and the samples were mounted in AF-1 antifade medium and viewed using a Zeiss LSM 510 confocal scanning microscope equipped with a c-apochromat 63×/1.2 water immersion objective (Carl Zeiss, Jena, Germany). Isotype- and concentration-matched control antibodies yielded consistently negative results. Images were analyzed using LSM 510 software (version 2.3; Carl Zeiss).

Detection of BLyS by Western blotting

Proteins from freshly isolated neutrophils were precipitated by 5 minutes’ incubation with 10% (weight/volume) trichloroacetic acid (TCA). Briefly, the neutrophil pellet was precipitated with ice-cold 10% TCA, and the precipitated proteins were spun down at 14,000g for 5 minutes at 4°C. The precipitate was washed 3 times in ice-cold acetone and taken up in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Proteins were separated on 12% SDS-PAGE gels. The equivalent of 3 × 106 cells per lane was loaded onto standard SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes. Western blots were blocked with 4% nonfat dry milk and developed with rat anti-BLyS antibody (Buffy-2; Axxora, Nottingham, UK). Blots were developed using an enhanced chemiluminescence method (Amersham Pharmacia, Buckinghamshire, UK).

Statistical analysis

The results presented are representative of a minimum of 3 experiments and, where appropriate, are expressed as the mean ± SD. Statistical significance was assessed by Student’s 2-tailed t-test or, for experiments using clinical samples, by nonparametric Mann-Whitney U test. P values less than 0.05 were considered significant.

RESULTS

Expression of BLyS by neutrophils from healthy donors

In dendritic cells and monocytes, BLyS is released from the cell surface by cleavage of the membrane-bound pro-form (12). Previous findings suggested that although neutrophils could produce and secrete BLyS, it could only be found in Golgi-associated intracellular compartments after priming for 20 hours with G-CSF (5,21). To clarify this unusual distribution of BLyS, neutrophils, monocytes, EBV-transformed B cells, and the promyelocytic cell line HL-60 were labeled with anti-BLyS antibody and analyzed by flow cytometry. HL-60 cells were used as positive controls, and an EBV-transformed B cell line was used as a negative control.

These experiments unequivocally showed expression of BLyS on the neutrophil surface (Figure 1A). To test whether this expression of BLyS was induced by the neutrophil purification protocol, we compared the level of BLyS expression on neutrophils stained in heparinized whole blood with that on neutrophils purified by Percoll gradient centrifugation. The results (not shown) indicated that the neutrophil isolation procedure did not affect the findings of neutrophil staining.

Figure 1.

Figure 1

Open in a new tab

Subcellular distribution of B lymphocyte stimulator (BLyS) in neutrophils. A, BLyS expression on the surface of neutrophils, monocytes, and HL-60 cells, detected by staining with a mouse monoclonal antibody. No staining was detected on Epstein-Barr virus–transformed B cells. All experiments were controlled with concentration-, species-, and isotype-matched irrelevant antibodies (shaded areas). B, Western blots of freshly isolated neutrophils, showing both the unprocessed 32-kd form and the processed 17-kd form of BLyS. C, Specificity of BLyS staining, as confirmed by blocking with recombinant (rec.) human BLyS. Irrel Ab = irrelevant antibody. D, Detection of both intracellular and extracellular pools of BLyS by immunofluorescence. BLyS expression was detected on the surface by staining of neutrophils in suspension with a mouse anti-BLyS antibody (green). Subsequent fixation, permeabilization, and staining with a mouse anti-BLyS monoclonal antibody (red) revealed both the extracellular and the granular intracellular pools of BLyS. The histograms in A and C show representative results from 1 of 3 independent experiments, with open areas indicating expression of BLyS.

Intracellular stores of BLyS have been shown to be processed to a 17-kd form that does not contain the transmembrane region (5). Western blots of lysates from freshly isolated neutrophils probed with anti-BLyS antibody showed bands at 17 kd and 32 kd (Figure 1B). The 32-kd band was consistent with the presence of membrane-bound BLyS, and the 17-kd form was consistent with the processed form stored in Golgi-related vesicles (5). As shown in Figure 1C, the specificity of BLyS staining was confirmed by the results of preincubation with anti-BLyS antibody and recombinant human BLyS.

To investigate the concomitant expression of membrane-bound BLyS and intracellular BLyS on neutrophils, the subcellular localization of BLyS was assayed by immunofluorescence staining and confocal microscopy. Externally accessible, membrane-bound BLyS was labeled by staining neutrophils with a mouse anti-BLyS antibody in suspension (visualized as green fluorescence in Figure 1D). Cells were then fixed, permeabilized, and colabeled with a mouse anti-BLyS antibody (visualized as red fluorescence in Figure 1D) to detect the release of BLyS both intracellularly and extracellularly.

The green surface staining of the neutrophils (Figure 1D) was consistent with our flow cytometric demonstration of BLyS on the cell surface. The distinct intracellular granular staining detected suggests that in addition to formation of a surface-bound pool of BLyS, neutrophils also express BLyS intracellularly. This detection of a preformed pool of intracellular BLyS is consistent with previous observations (5). However, expression of membrane-bound BLyS on neutrophils has not been described before, but is consistent with the localization that has been described for other myeloid cells. Our observations confirm that BLyS is expressed both on the surface and in intracellular stores of freshly isolated human neutrophils.

Expression of BLyS on peripheral blood and synovial fluid neutrophils

We compared the level of BLyS expression on neutrophils isolated from the peripheral blood and synovial fluid of patients with RA with the levels found on neutrophils isolated from the peripheral blood and synovial fluid of healthy controls. As shown in Figure 2, neutrophils from the synovial fluid expressed significantly lower levels of BLyS on their surface when compared with the expression on peripheral blood neutrophils, from either RA patients or healthy controls. Among the patients tested, we found no influence of disease duration or drug regimen on the expression of BLyS on the neutrophil surface. These findings led us to investigate how the microenvironment of inflammation activates shedding mechanisms to release biologically active BLyS from the surface of neutrophils and thus contribute to B cell autoimmunity.

Figure 2.

Figure 2

Open in a new tab

Expression of B lymphocyte stimulator (BLyS) on neutrophils from the blood and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and healthy controls. Neutrophils were stained for BLyS expression, and stained samples were analyzed by flow cytometry. Results are the mean fluorescence intensity of BLyS staining. Bars indicate the median. * = P < 0.05.

Induction of release of the membrane-bound pool of BLyS by proinflammatory cytokines

Incubation of blood neutrophils with proinflammatory cytokines for 1 hour showed that treatment with TNFα led to a dramatic decrease in BLyS expression on the neutrophil surface (Figures 3A and B). In the same experiments, the level of BLyS detectable in the supernatant was increased when neutrophils were incubated with TNFα, suggesting that TNFα may activate release of BLyS directly from the cell surface (Figure 3C). In addition, GM-CSF and IL-8 had a minor effect on BLyS release. Incubation of neutrophils with the same selection of cytokines for 18 hours showed that over a longer incubation period, both G-CSF and TNFα induced release of BLyS into the supernatant (Figure 3E).

Figure 3.

Figure 3

Open in a new tab

Effects of stimulation with a range of proinflammatory cytokines on extracellular and intracellular release of B lymphocyte stimulator (BLyS) from neutrophils. BLyS expression was assessed on the surface of neutrophils after short-term (1-hour) incubation with cytokines, by flow cytometry analysis (A) and quantification of BLyS staining (B). Open areas in A indicate BLyS expression. The expression of BLyS was also quantified after short-term (1-hour) incubation with cytokines in the supernatant (C). Expression of BLyS on the surface of neutrophils (D) and in the supernatant (E) was further assessed in long-term (18-hour) cultures with cytokines. Tumor necrosis factor α (TNFα)–induced loss of membrane-expressed BLyS was assessed in time-course analyses (F); the maximum change in BLyS expression was observed within 30 minutes. For determination of maximal TNFα-induced BLyS release, concentrations of TNFα were tested in titration experiments, showing that BLyS release could be triggered by physiologically relevant concentrations of TNFα (G). CD95 expression by neutrophils (H) and BLyS expression by monocytes (I) after 1-hour incubation with TNFα were also assessed. Results in B–G are the mean and SD of 3 separate experiments, expressed as the percent of mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. In H and I, shaded areas represent staining with irrelevant control antibody (irrel. ab.), while open areas represent CD95 staining of control neutrophils (dotted line) and TNFα-treated neutrophils (solid line) (H) or BLyS staining of control monocytes (dotted line) and TNFα-treated monocytes (solid line) (I). * = P < 0.05 versus control. G-CSF = granulocyte colony-stimulating factor; IL-1β = interleukin-1β; IFNβ = interferon-β; GM-CSF = granulocyte–macrophage CSF.

Interestingly, the release of BLyS induced by G-CSF was not associated with changes in the levels of BLyS expressed on the membrane (Figure 3D), implying that the G-CSF–induced BLyS release was from intracellular stores. In contrast, the release of BLyS induced by TNFα was much more rapid and was accompanied by the loss of membrane-associated BLyS (Figure 3D), as seen after 1 hour and persisting for at least 18 hours.

Time-course experiments showed that TNFα induced rapid release of BLyS from the cell surface (Figure 3F). Titration experiments revealed that release of BLyS was induced at physiologically relevant concentrations of TNFα (Figure 3G). As shown in Figure 3H, CD95 staining of neutrophils was not affected by TNFα treatment, suggesting that the TNFα-induced loss of BLyS expression on neutrophils was specific for BLyS. The level of BLyS expression on monocytes was not affected by incubation with TNFα ( Figure 3I), suggesting that the release of BLyS is a neutrophil-specific mechanism.

Thus, the observed expression of BLyS from intracellular stores following prolonged incubation with G-CSF is consistent with previous findings (5,21). In addition, in the present study, we were able to demonstrate constitutive expression of BLyS by neutrophils, and induction of the release of membrane-bound BLyS by TNFα.

Characterization of the proteolytic activity mediating TNFα-dependent BLyS release

TNF family members are, in most cases, released from the cell surface by proteolytic enzymes (20). These include metalloproteases, such as ADAM-17/TACE, as well as serine proteases, such as proteinase 3, cathepsin G, elastase, and furin (12,16). Inhibitors of both groups of proteases were used to explore the mechanism of TNFα-induced BLyS release in neutrophils from the peripheral blood (Figure 4A).

Figure 4.

Figure 4

Open in a new tab

Dependence of tumor necrosis factor α (TNFα)–activated release of B lymphocyte stimulator (BLyS) on serine protease activity, and involvement of membrane translocation and activation of furin. A and B, Neutrophils were preincubated for 30 minutes with a range of inhibitors and then cultured in the absence or presence of TNFα. A, The metalloprotease inhibitor GW280264X (GW), for metalloproteases ADAM-17 and ADAM-10, did not affect TNFα-induced BLyS release from the neutrophil membrane. Neutrophils pretreated with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) did not show surface release of BLyS. Neutrophils pretreated with specific inhibitors for cathepsin G (CGI), proteinase 3 (PRI), and elastase (EI) showed release of BLyS if stimulated with TNFα. A specific inhibitor of furin (FCI) blocked loss of BLyS from the neutrophil membrane. Results are the percent of mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. B, FCI also blocked release of BLyS into the supernatant in both the presence and absence of TNFα. C and D, To determine furin expression, neutrophils were incubated with or without TNFα for 15 minutes and labeled with an anti-furin antibody. Values in A, B, and D are the mean and SD of 3 separate experiments. The histogram in C shows representative results from 3 independent experiments, with open areas indicating furin expression. * = P < 0.05 versus control.

The hydroxamate GW280264X can block TACE and other metalloproteases of the disintegrin family (22). However, there was no effect of this inhibitor on the release of BLyS (Figure 4A), which suggests that there is no role for this group of proteases in the expression of BLyS from neutrophils. Incubation with AEBSF, a general serine protease inhibitor, blocked BLyS release (Figure 4A). Given the efficient blockade of BLyS release by AEBSF, obvious candidates for the BLyS-releasing activity were the 3 major serine proteases (elastase, cathepsin G, and proteinase 3) (23,24) that are released to the neutrophil membrane after stimulation. Inhibitors of these proteases failed to inhibit BLyS release (Figure 4A).

Another serine protease, furin, has been implicated in the release of BLyS from dendritic cells (12). The members of this family of pro-protein convertases are calcium-dependent serine proteases that normally reside in the nearby Golgi complex. They can, however, also shuttle between the Golgi complex and the plasma membrane (25). Figures 4A and B show that a specific, cell-permeable furin convertase inhibitor (FCI) abrogated the TNFα-induced BLyS loss from the neutrophil membrane as well as the TNFα-induced increase in BLyS levels in the supernatant. Furthermore, TNFα treatment led to an increased level of furin expression on the membrane of blood-derived neutrophils (Figures 4C and D).

RA synovial fluid–activated release of BLyS in a TNFα-dependent manner

The results presented thus far suggest that TNFα activates shedding of BLyS from the surface of neutrophils by a mechanism involving activation of furin convertase. Furthermore, neutrophils purified from RA synovial fluid express low levels of BLyS on their surface. Concentrations of TNFα that have been reported to be present in RA synovial fluid range up to ~90 pg/ml (26). The results of our titration experiments suggested that TNFα concentrations of ~10 pg/ml are sufficient to activate BLyS shedding, and that this level can be easily reached in the synovial fluid of RA patients. These calculations, however, do not take into consideration that synovial fluid contains a multitude of other factors, such as other cytokines and soluble TNF receptors, that might counteract or amplify the effect of TNFα on BLyS release.

We therefore incubated neutrophils isolated from the peripheral blood of healthy donors with the synovial fluid from RA patients. As shown in Figure 5, incubation with RA synovial fluid induced the loss of BLyS from the cell surface of neutrophils. Blocking of TNFα using a neutralizing antibody significantly inhibited this effect. Based on these observations, we conclude that TNFα in the synovial microenvironment activates the furin-induced cleavage of membrane-bound BLyS from neutrophils.

Figure 5.

Figure 5

Open in a new tab

Rheumatoid arthritis (RA) synovial fluid (SF)–activated release of B lymphocyte stimulator (BLyS) in a tumor necrosis factor α (TNFα)–dependent manner. Neutrophils were purified from the blood of healthy donors, exposed to 50% SF from RA patients for 1 hour, and stained for BLyS expression. Compared with control cultures, cultures with SF showed a drop in BLyS expression on the surface of blood neutrophils. Neutralizing anti-TNFα antibodies (SF + nAb) significantly reduced this effect. Neutralizing antibodies on their own (nAb) had no significant influence on BLyS expression. To test the efficiency of the neutralizing antibody, we cultured neutrophils with TNFα alone, and blocked its effect with the neutralizing antibody (TNFα + nAb). Results are the mean fluorescence intensity (MFI) of BLyS staining, with the MFI of BLyS staining of untreated neutrophils defined as 100%. Bars indicate the median. * = P < 0.05.

DISCUSSION

BLyS is an important regulator of B cell autoimmunity. Significant levels of soluble BLyS are found in the serum and synovial fluid of patients with RA (15). In the present study, we have shown that neutrophils, which are the dominant cell population in synovial fluid, constitutively express BLyS. We have identified TNFα as a potent and effective microenvironmental factor that can rapidly activate the release of membrane-bound BLyS. Titration experiments showed that a significant loss of membrane-bound BLyS can be induced by TNFα concentrations as low as 10 pg. This concentration is well in the range of concentrations determined by McNearney et al (26). As confirmation, synovial fluid from patients with RA was able to induce BLyS release in a TNFα-dependent manner. The low ex vivo expression of BLyS detected on synovial neutrophils would therefore be consistent with a model in which neutrophils shed BLyS as they enter the rheumatoid joint and encounter TNFα.

Recent developments in the treatment of RA patients with B cell–targeting drugs (27) show that the role of B cells in the pathophysiology of RA was previously underestimated. Ectopic sites of B cell differentiation and hypermutation have been identified in the rheumatoid synovium. In addition, we recently found that the very early phase of RA is associated with high levels of IL-4 and IL-13, cytokines that are mostly associated with B cell responses supported by Th2 cells (28). These observations suggest a key role of B cell stimulation in the very early stages of RA. Moreover, the factors involved in autoimmune B cell activation or survival may represent rational therapeutic targets that could halt progression of the disease.

BLyS is therefore an attractive therapeutic target, although initial findings from a phase II clinical trial in which blocking monoclonal antibodies against BLyS were used showed only modest clinical improvements (29). However, a recent study showed that in patients undergoing B cell depletion therapy with rituximab, the BLyS levels increased in direct relation to the drop in B cell numbers (30). It is likely that increased levels of BLyS contribute to the reemergence of B cells observed within months of B cell depletion therapy. This raises the possibility that combining anti-BLyS therapy with B cell depletion therapy may increase the duration of B cell depletion.

Thus far, dendritic cells, macrophages, and synoviocytes were considered to be the main sources of BLyS in the rheumatoid joint. Indeed, the results of a recent study suggested that macrophages are the major source of BLyS production in synovial tissue in the established phase of RA (31). However, that study did not consider cells within the synovial fluid or factors that might be involved in the early phases of the development of RA prior to the development of pannus hyperplasia. In this context, Tan et al (32) showed that the concentration of BLyS in synovial fluid correlated positively with the absolute numbers of both monocytes and neutrophils. New data from our group and other investigators (5,21) have shown that neutrophils can produce significant quantities of BLyS and therefore have the potential to influence B cell activation.

The results of the present study show that BLyS is expressed both on the surface of neutrophils and in preprocessed intracellular pools. We investigated the regulation of BLyS release by proinflammatory cytokines and found that among a range of neutrophil-stimulating cytokines, only TNFα was able to trigger BLyS release from the neutrophil membrane. In contrast, G-CSF increased release of BLyS from intracellular stores without changing the membrane level of BLyS. We conclude from these studies that freshly isolated neutrophils from normal healthy donors constitutively express BLyS at 2 separate sites, and that release from these stores is affected by different proinflammatory cytokines.

The membrane-associated pool of BLyS can be rapidly released by stimulation of neutrophils with TNFα. This release involves the translocation of the pro-protein convertase furin to the cell surface and subsequent shedding of BLyS. In contrast, the intracellular pool of BLyS is released from the cells by G-CSF treatment, which is in accord with previous findings (5,21), although in one of these studies, membrane-bound BLyS on neutrophils was not detected (5). It is likely that a difference in sensitivity of the staining methods used may account for these discrepancies. In the same study, the level of BLyS detected on the surface of HL-60 cells was very low, despite previous reports by other investigators of significant levels of BLyS on these cells (33).

BLyS is an essential component of B cell homeostasis, but its excess production results in multiple autoimmune symptoms. BLyS-transgenic mice have elevated serum immunoglobulin levels, including rheumatoid factor, as well as circulating immune complexes. BLyS-transgenic mice also show evidence of B cell hyperplasia (11,34,35). Of note, BLyS can potentially deviate the negative selection of B cells at the crucial T2 stage. These observations support a model of autoimmunity in these mice that is driven by excessive levels of BLyS, which generates a survival signal strong enough to overcome the death signals that are triggered by autoantigen binding to the B cell receptor (36).

The site of production of BLyS has been a topic of much debate. The concentration of BLyS in RA synovial fluid has been reported to be higher than the serum concentration of BLyS, suggesting that production occurs at the site of inflammation (15). In this context, the recent findings by Collins et al (37) are of interest, in that they demonstrated that messenger RNA expression of BLyS in blood leukocytes of patients with systemic lupus erythematosus (SLE) correlated better with disease severity than did the levels of BLyS in the blood. Those authors suggested that in SLE patients, but not in RA patients, blood leukocytes may be a major source of BLyS. Taken together, the evidence from the current literature suggests that depending on the underlying disease, increased production of BLyS can occur either systemically or locally at sites of inflammation. Our findings add to this body of evidence by showing that synovial fluid neutrophils are also a major source of BLyS.

The protease responsible for processing and shedding of BLyS in other myeloid cells has been proposed to be a furin-like convertase. However, in our experiments with neutrophils, it was important to rule out any role of other proteases known to be expressed on the surface of activated neutrophils (e.g., elastase, cathepsin G, and proteinase 3) (23,24). Specific inhibitors of elastase, cathepsin G, and proteinase 3 did not block the TNFα-mediated release of BLyS from the neutrophil surface, whereas a furin inhibitor was effective.

A furin-mediated mechanism of BLyS release was first proposed by Schneider et al (12). Their logic was initially based on sequence data, which showed that the stalk region of BLyS contains the polybasic target site for cleavage by furin-like convertases. Elimination of this target site by mutation of amino acids 132 and 133 at this polybasic cleavage site yielded a form of BLyS that is not released by cleavage (33).

A role for furin in the shedding of a surface-bound molecule is potentially difficult to propose, since the main site of activity for this group of enzymes lies in the processing of pro-proteins in the trans-Golgi network (17). However, recent observations of furin translocation to the plasma membrane support the possible involvement of this enzyme in the shedding of a type II transmembrane protein from the cell surface (25,38). A previous indication of its ability to cleave proteins from the cell membrane came from studies showing that cell surface–bound furin is needed for the full activation of anthrax toxin protective antigen (39). A complex pathway characterized by the shuttling of furin between several subcellular compartments has since been unravelled (40,41). The mechanisms by which treatment of neutrophils with TNFα leads to the translocation and activation of furin are currently not understood and will require further investigation.

We conclude from these studies that the stimulatory effects of TNFα lead to the release of BLyS from the surface of invading neutrophils at sites of inflammation. This release is mediated by activation of furin. In conditions involving massive influx of neutrophils into a limited space, such as the inflamed rheumatoid joint, BLyS released from neutrophils is likely to reach biologically significant levels and thus contribute to B cell autoimmunity.

ACKNOWLEDGMENT

We thank Mrs. Hema Chahal for expert technical assistance.

Drs. Assi and Wong’s work was supported by PhD studentship grants from the University of Birmingham, Birmingham, UK. Dr. Ludwig’s work was supported by the German Research Foundation’s Interdisciplinary Centre for Clinical Research (IZKF “Biomat”), RWTH Aachen University, Aaachen, Germany. Drs. Raza and Salmon’s work was supported by grants from the Arthritis Research Campaign, UK. Dr. Gordon’s work was supported by grants from the Arthritis Research Campaign, UK and Lupus UK. Dr. Lord’s work was supported by grants from the Arthritis Research Campaign, UK and the European Union’s Sixth Framework Programme for Research and Technological Development. Dr. Scheel-Toellner is recipient of a Non-Clinical Career Development Fellowship from the Arthritis Research Campaign, UK; her work was also supported by a grant from the University of Birmingham, Birmingham, UK.

REFERENCES

  • 1.Pillinger MH, Abramson SB. The neutrophil in rheumatoid arthritis. Rheum Dis Clin North Am. 1995;21:691–714. [PubMed] [Google Scholar]
  • 2.Chen M, Lam BK, Kanaoka Y, Nigrovic PA, Audoly LP, Austen KF, et al. Neutrophil-derived leukotriene B4 is required for inflammatory arthritis. J Exp Med. 2006;203:837–42. doi: 10.1084/jem.20052371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Wipke BT, Allen PM. Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis. J Immunol. 2001;167:1601–8. doi: 10.4049/jimmunol.167.3.1601. [DOI] [PubMed] [Google Scholar]
  • 4.Tanaka D, Kagari T, Doi H, Shimozato T. Essential role of neutrophils in anti-type II collagen antibody and lipopolysaccharide-induced arthritis. Immunology. 2006;119:195–202. doi: 10.1111/j.1365-2567.2006.02424.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Scapini P, Nardelli B, Nadali G, Calzetti F, Pizzolo G, Montecucco C, et al. G-CSF-stimulated neutrophils are a prominent source of functional BLyS. J Exp Med. 2003;197:297–302. doi: 10.1084/jem.20021343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Nell VP, Machold KP, Stamm TA, Eberl G, Heinzl H, Uffmann M, et al. Autoantibody profiling as early diagnostic and prognostic tool for rheumatoid arthritis. Ann Rheum Dis. 2005;64:1731–6. doi: 10.1136/ard.2005.035691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Raza K, Breese M, Nightingale P, Kumar K, Potter T, Carruthers DM, et al. Predictive value of antibodies to cyclic citrullinated peptide in patients with very early inflammatory arthritis. J Rheumatol. 2005;32:231–8. [PMC free article] [PubMed] [Google Scholar]
  • 8.Shu HB, Hu WH, Johnson H. TALL-1 is a novel member of the TNF family that is down-regulated by mitogens. J Leukoc Biol. 1999;65:680–3. [PubMed] [Google Scholar]
  • 9.Mukhopadhyay A, Ni J, Zhai Y, Yu GL, Aggarwal BB. Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-κB, and c-Jun NH2-terminal kinase. J Biol Chem. 1999;274:15978–81. doi: 10.1074/jbc.274.23.15978. [DOI] [PubMed] [Google Scholar]
  • 10.Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, et al. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science. 1999;285:260–3. doi: 10.1126/science.285.5425.260. [DOI] [PubMed] [Google Scholar]
  • 11.Gross JA, Johnston J, Mudri S, Enselman R, Dillon SR, Madden K, et al. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature. 2000;404:995–9. doi: 10.1038/35010115. [DOI] [PubMed] [Google Scholar]
  • 12.Schneider P, MacKay F, Steiner V, Hofmann K, Bodmer JL, Holler N, et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med. 1999;189:1747–56. doi: 10.1084/jem.189.11.1747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Groom J, Kalled SL, Cutler AH, Olson C, Woodcock SA, Schneider P, et al. Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren’s syndrome. J Clin Invest. 2002;109:59–68. doi: 10.1172/JCI14121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zhang J, Roschke V, Baker KP, Wang Z, Alarcon GS, Fessler BJ, et al. Cutting edge: a role for B lymphocyte stimulator in systemic lupus erythematosus. J Immunol. 2001;166:6–10. doi: 10.4049/jimmunol.166.1.6. [DOI] [PubMed] [Google Scholar]
  • 15.Cheema GS, Roschke V, Hilbert DM, Stohl W. Elevated serum B lymphocyte stimulator levels in patients with systemic immune–based rheumatic diseases. Arthritis Rheum. 2001;44:1313–9. doi: 10.1002/1529-0131(200106)44:6<1313::AID-ART223>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  • 16.Coeshott C, Ohnemus C, Pilyavskaya A, Ross S, Wieczorek M, Kroona H, et al. Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc Natl Acad SciUSA. 1999;96:6261–6. doi: 10.1073/pnas.96.11.6261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Nakayama K. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J. 1997;327:625–35. doi: 10.1042/bj3270625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315–24. doi: 10.1002/art.1780310302. [DOI] [PubMed] [Google Scholar]
  • 19.De Boer M, Roos R. Metabolic comparison between basophils and other leukocytes from human blood. J Immunol. 1986;136:3447–54. [PubMed] [Google Scholar]
  • 20.Gearing AJ, Beckett P, Christodoulou M, Churchill M, Clements J, Davidson AH, et al. Processing of tumour necrosis factor-alpha precursor by metalloproteinases. Nature. 1994;370:55–7. doi: 10.1038/370555a0. [DOI] [PubMed] [Google Scholar]
  • 21.Scapini P, Carletto A, Nardelli B, Calzetti F, Roschke V, Merigo F, et al. Proinflammatory mediators elicit secretion of the intracellular B-lymphocyte stimulator pool (BLyS) that is stored in activated neutrophils: implications for inflammatory diseases. Blood. 2005;105:830–7. doi: 10.1182/blood-2004-02-0564. [DOI] [PubMed] [Google Scholar]
  • 22.Hundhausen C, Misztela D, Berkhout TA, Broadway N, Saftig P, Reiss K, et al. The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion. Blood. 2003;102:1186–95. doi: 10.1182/blood-2002-12-3775. [DOI] [PubMed] [Google Scholar]
  • 23.Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol. 1995;131:775–89. doi: 10.1083/jcb.131.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Campbell EJ, Campbell MA, Owen CA. Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition. J Immunol. 2000;165:3366–74. doi: 10.4049/jimmunol.165.6.3366. [DOI] [PubMed] [Google Scholar]
  • 25.Thomas G. Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol. 2002;3:753–66. doi: 10.1038/nrm934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.McNearney T, Baethge BA, Cao S, Alam R, Lisse JR, Westlund KN. Excitatory amino acids, TNF-α, and chemokine levels in synovial fluids of patients with active arthropathies. Clin Exp Immunol. 2004;137:621–7. doi: 10.1111/j.1365-2249.2004.02563.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Edwards JC, Cambridge G. B-cell targeting in rheumatoid arthritis and other autoimmune diseases. Nat Rev Immunol. 2006;6:394–403. doi: 10.1038/nri1838. [DOI] [PubMed] [Google Scholar]
  • 28.Raza K, Falciani F, Curnow SJ, Ross EJ, Lee CY, Akbar AN, et al. Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin. Arthritis Res Ther. 2005;7:R784–95. doi: 10.1186/ar1733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ding C, Jones G. Belimumab Human Genome Sciences/Cambridge Antibody Technology/GlaxoSmithKline. Curr Opin Investig Drugs. 2006;7:464–72. [PubMed] [Google Scholar]
  • 30.Cambridge G, Stohl W, Leandro MJ, Migone TS, Hilbert DM, Edwards JC. Circulating levels of B lymphocyte stimulator in patients with rheumatoid arthritis following rituximab treatment: relationships with B cell depletion, circulating antibodies, and clinical relapse. Arthritis Rheum. 2006;54:723–32. doi: 10.1002/art.21650. [DOI] [PubMed] [Google Scholar]
  • 31.Seyler TM, Park YW, Takemura S, Bram RJ, Kurtin PJ, Goronzy JJ, et al. BLyS and APRIL in rheumatoid arthritis. J Clin Invest. 2005;115:3083–92. doi: 10.1172/JCI25265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tan SM, Xu D, Roschke V, Perry JW, Arkfeld DG, Ehresmann GR, et al. Local production of B lymphocyte stimulator protein and APRIL in arthritic joints of patients with inflammatory arthritis. Arthritis Rheum. 2003;48:982–92. doi: 10.1002/art.10860. [DOI] [PubMed] [Google Scholar]
  • 33.Nardelli B, Belvedere O, Roschke V, Moore PA, Olsen HS, Migone TS, et al. Synthesis and release of B-lymphocyte stimulator from myeloid cells. Blood. 2001;97:198–204. doi: 10.1182/blood.v97.1.198. [DOI] [PubMed] [Google Scholar]
  • 34.Mackay F, Woodcock SA, Lawton P, Ambrose C, Baetscher M, Schneider P, et al. Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J Exp Med. 1999;190:1697–710. doi: 10.1084/jem.190.11.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Khare SD, Sarosi I, Xia XZ, McCabe S, Miner K, Solovyev I, et al. Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice. Proc Natl Acad SciUSA. 2000;97:3370–5. doi: 10.1073/pnas.050580697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Mackay F, Mackay CR. The role of BAFF in B-cell maturation, T-cell activation and autoimmunity. Trends Immunol. 2002;23:113–5. doi: 10.1016/s1471-4906(01)02159-7. [DOI] [PubMed] [Google Scholar]
  • 37.Collins CE, Gavin AL, Migone TS, Hilbert DM, Nemazee D, Stohl W. B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels. Arthritis Res Ther. 2006;8:R6. doi: 10.1186/ar1855. [online only] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Crump CM, Xiang Y, Thomas L, Gu F, Austin C, Tooze SA, et al. PACS-1 binding to adaptors is required for acidic cluster motifmediated protein traffic. EMBO J. 2001;20:2191–201. doi: 10.1093/emboj/20.9.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Klimpel KR, Molloy SS, Thomas G, Leppla SH. Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc Natl Acad SciUSA. 1992;89:10277–81. doi: 10.1073/pnas.89.21.10277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Mayer G, Boileau G, Bendayan M. Sorting of furin in polarized epithelial and endothelial cells: expression beyond the Golgi apparatus. J Histochem Cytochem. 2004;52:567–79. doi: 10.1177/002215540405200502. [DOI] [PubMed] [Google Scholar]
  • 41.Molloy SS, Thomas L, VanSlyke JK, Stenberg PE, Thomas G. Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J. 1994;13:18–33. doi: 10.1002/j.1460-2075.1994.tb06231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES