Sylvie Berthier

Human B lymphocytes synthesize the 92-kDa gelatinase, matrix metalloproteinase-9

Matrix metalloproteinases (MMPs) are involved in the remodeling of connective tissue as well as in disease states associated with acute and chronic inflammation or tumoral metastatic processes. Despite detailed and extensive studies of the mechanisms of lymphocyte extravasation, remarkably little is known about the expression and regulation of metalloproteinases involved in the migratory process. By using zymography and reverse transcription-polymerase chain reaction experiments, we have demonstrated that Epstein-Barr virus-immortalized B lymphocytes are able to secrete a 92-kDa metalloproteinase with gelatinolytic activity which has been purified and identified as being MMP-9. Moreover, the tissue inhibitor of metalloproteinase was shown to be constitutively expressed by the B cells. The expression of 92-kDa gelatinase is mediated by cytokines, growth factors, lipopolysaccharide, concanavalin A, and the tumor promotor phorbol 12-myristate 13-acetate. Time dependence activity increased rapidly up to 24 h of incubation with lipopolysaccharide or concanavalin A stimulation while it requires a delay and more time to have an optimum effect when cytokines were the stimulating agents; transforming growth factor-β abolished 92-kDa gelatinase production. Both staurosporine and wortmannin are inductive stimuli, and the level of MMP-9 secreted into the media is greater than that observed with other agents except concanavalin A. Elicitation of the chemotactic migration of B cells through a model basement membrane by lipopolysaccharide was shown to be correlated with gelatinase expression and inhibited by 7 mm captopril. Our study indicates that Epstein-Barr virus-B lymphocytes express 92-kDa gelatinase, the production of which can be modified by a variety of physiological and pharmacological signals which have been shown to differ according to the cell type.

Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases

OBJECTIVE We investigated SF and serum proteomic fingerprints of patients suffering from RA, OA and other miscellaneous inflammatory arthritides (MIAs) in order to identify RA-specific biomarkers. METHODS SF profiles of 65 patients and serum profiles of 31 patients were studied by surface-enhanced laser desorption and ionization-time-of-flight-mass spectrometry technology. The most discriminating RA biomarkers were identified by matrix-assisted laser desorption ionization-time of flight and their overexpression was confirmed by western blotting and ELISA. RESULTS Three biomarkers of 10 839, 10 445 and 13 338 Da, characterized as S100A8, S100A12 and S100A9 proteins, were the most up-regulated proteins in RA SF. Their expression was about 10-fold higher in RA SF vs OA SF. S100A8 exhibited a sensitivity of 82% and a specificity of 69% in discriminating RA from other MIAs, whereas S100A12 displayed a sensitivity of 79% and a specificity of 64%. Three peptides of 3351, 3423 and 3465 Da, corresponding to the alpha-defensins-1, -2 and -3, were also shown to differentiate RA from other MIAs with weaker sensitivity and specificity. Levels of S100A12, S100A8 and S100A9 were statistically correlated with the neutrophil count in MIA SF but not in the SF of RA patients. S100A8, S100A9, S100A12 and alpha-defensin expression in serum was not different in the three populations. CONCLUSION The most enhanced proteins in RA SF, the S100A8, S100A9 and S00A12 proteins, distinguished RA from MIA with high accuracy. Possible implication of resident cells in this increase may play a role in RA physiopathology.

Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states.

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67‐phox, p47‐phox, p40‐phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemopro‐tein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphi‐phile agent with a maximum turnover (125 mol O2−•s−1τnol heme b−1), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an “open conformation,” able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (~50 mol O2− •s−1 Triol heme b−1). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67‐phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully “open” cyto‐chrome b558 conformation. Moreover, their effect was independent of p67‐phox. Data point out two potential cytochrome b558 activation states.—Paclet, M‐H., Berthier, S., Kuhn, L., Garin, J., Morel, F. Regulation of phagocyte NADPH oxidase activity: identification of two cytochrome b558 activation states. FASEB J. 21, 1244–1255 (2007)

TIMP-1/MMP-9 imbalance in an EBV-immortalized B lymphocyte cellular model: evidence for TIMP-1 multifunctional properties.

Tissue inhibitors of metalloproteinases (TIMPs) were initially described as agents controlling metalloproteinase activity. The purpose of this study was to investigate the expression and the roles of TIMP-1 secreted by Epstein-Barr-virus (EBV)-immortalized B lymphocytes. TIMP-1 was isolated from conditioned medium of interleukin (IL)-1beta stimulated EBV-B lymphocytes; purified TIMP-1 was identified by mass spectrometry and immunochemistry. TIMP-1-free MMP-9 was quantified after purification by zymography and enzyme-linked immunosorbent assay. EBV-B lymphocyte-secreted TIMP-1 inhibited MMP-9 gelatinolytic activity resulting in decreased B-cell transmigration as measured in vitro. The release of huge amounts of TIMP-1 in proportion to MMP-9 from B lymphocytes after EBV transformation was shown to be correlated with secretion of IL-10 and dependent on culture time. In contrast, there was little TIMP-1 and almost no IL-10 released from native B cells, suggesting a possible IL-10 mediated autocrine regulation mechanism of TIMP-1 synthesis. The MMP-9/TIMP-1 imbalance observed in the culture medium of EBV-B lymphocytes (TIMP-1>MMP-9) and of native B cells (MMP-9>TIMP-1) is suggestive of a new function for TIMP-1. We propose that TIMP-1 acts as a survival factor controlling B-cell growth and apoptosis through an autocrine regulation process involving IL-10 secreted by EBV-B lymphocytes.

Further characterization of the gelatinase-containing particles of human neutrophils.

In addition to azurophil and specific granules, a third storage compartment is known to exist in the neutrophils. This compartment which consists of morphologically heterogeneous particles is characterized by a high specific activity in gelatinase. A gelatinase enriched fraction was prepared by subcellular fractionation of neutrophil homogenates using rate zonal centrifugation. This fraction was enriched in diamine oxidase. Among the proteins released from the neutrophils upon stimulation by formyl peptides, those belonging to the gelatinase enriched fraction were determined after removal of the proteins from specific and azurophil granules by selective immunoadsorption. Gelatinase was recovered together with tetranectin, beta 2-microglobulin and diamine oxidase in the same fraction. Differences in the kinetics of release of gelatinase and diamine oxidase versus vitamin B-12-binding protein suggest that the proteins belong to distinct subcellular structures.

How Important are S100A8/S100A9 Calcium Binding Proteins for the Activation of Phagocyte NADPH Oxidase, Nox2

S100A8 and S100A9 are two soluble calcium-binding proteins highly expressed in myeloid cells, mainly neutrophils (45% of cytosolic proteins) or monocytes (1-5%) and also early differentiated macrophages. In neutrophils, they are believed to be expressed as a 1/1 non covalent heterodimer; the process of dimer and mainly tetramer formation is calcium dependent. The S100A8/S100A9 calcium loaded complex binds arachidonic acid and shuttles between cytosol and plasma membrane upon neutrophil stimulation. Neutrophils display, upon stimulation, a respiratory burst in which the cells catalyze NADPH oxidase activity through a redox membrane hemoprotein, cytochrome b558, which is constituted of 2 subunits: gp91-phox, the redox core and p22- phox the stabilizing partner. In neutrophils, this activity is transitory: to be active, regulatory cytosolic factors, p67-phox, p47-phox, p40-phox and Rac1/2 assemble with membrane cytochrome b558. Both S100A8 and S100A9 were recently introduced as partners for NADPH oxidase activation and associate with the cytosolic activating factors especially p67-phox and Rac1/2. Moreover, S100A8/S100A9 potentiates NADPH oxidase activity. This was observed ex vivo after co-transfection of genes encoding both S100A8 and S100A9 in B lymphocytes that express all the components of the phagocyte oxidase, but display a very low NADPH oxidase activity (in these cells, S100A8 and S100A9 are not present endogenously). In the biological function of S100A8/S100A9, S100A8 is a strategic protein that needs to be active in vivo as in vitro, its specific partner S100A9. New data introduce S100A8 and S100A9 as positive effectors in allosteric regulation of phagocyte NADPH oxidase activity.

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6‐phosphofructo‐2‐kinase (PFK‐2). The purpose of this workwas to study the relationship between PFK‐2 and NADPHoxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK‐2 with Nox2 complex in stimulated neutrophils. In its active form, PFK‐2 catalyzes the production of fructose‐2, 6‐bisphosphate, which is the main allosteric activator of phosphofructo‐1‐kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK‐2 phosphorylation and cell depletion in PFK‐2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energeticmetabolismincrease thatwas induced by inflammatory conditions. PFK‐2 seems to be a strategic element that links NADPH oxidase activation and glycolysismodulation, and, as such, is proposedas a potential therapeutic target in inflammatory diseases.—Baillet, A., Hograindleur, M.‐A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.‐H. Unexpected function of the phagocyteNADPHoxidase in supporting hyperglycolysis in stimulated neutrophils:key role of 6‐phosphofructo‐2‐ kinase. FASEB J. 31, 663–673 (2017). www.fasebj.org

Regulation of TIMP-1 phenotypic expression in Epstein–Barr virus-immortalized B lymphocytes

Normal B lymphocytes as well as malignant B cells extravasate from blood circulation during physiological and pathological processes and require matrix metalloproteinases (MMPs) to facilitate trafficking through the subendothelial basal lamina and the extracellular matrix. We have previously shown that Epstein-Barr virus (EBV)-immortalized B lymphocytes constitutively synthesized low levels of MMP-9 and huge amounts of its preferential inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). In the present study, TIMP-1 phenotypic expression was extensively investigated in response to various mediators including interleukins, chemokines, growth factors and tumor promotor, and was compared to MMP-9 synthesis. Results showed a roughly constitutive TIMP-1 expression opposed to an inducible MMP-9 synthesis. Nevertheless, further analysis of TIMP-1 synthesis showed the existence of regulation mechanisms: modulation of intracellular Ca(2+) concentration as well as cation ionophore monensin were demonstrated to influence TIMP-1 production and secretion. The precise pathways implicated in these regulation mechanisms are currently under survey.

Anticancer properties of sodium selenite in human glioblastoma cell cluster spheroids

Glioblastoma (GBM) is the most common type of primary tumor of the central nervous system with a poor prognosis, needing the development of new therapeutic drugs. Few studies focused on sodium selenite (SS) effects in cancer cells cultured as multicellular tumor spheroids (MCTS or 3D) closer to in vivo tumor. We investigated SS anticancer effects in three human GBM cell lines cultured in 3D: LN229, U87 (O(6)-methyguanine-DNA-methyltransferase (MGMT) negative) and T98G (MGMT positive). SS absorption was evaluated and the cytotoxicity of SS and temozolomide (TMZ), the standard drug used against GBM, were compared. SS impacts on proliferation, cell death, and invasiveness were evaluated as well as epigenetic modifications by focusing on histone deacetylase (HDAC) activity and dimethyl-histone-3-lysine-9 methylation (H3K9m2), after 24h to 72h SS exposition. SS was absorbed by spheroids and was more cytotoxic than TMZ (i.e., for LN229, the IC50 was 38 fold-more elevated for TMZ than SS, at 72h). SS induced a cell cycle arrest in the S phase and apoptosis via caspase-3. SS decreased carbonic anhydrase-9 (CA9) expression, invasion on a Matrigel matrix and modulated E- and N-Cadherin transcript expressions. SS decreased HDAC activity and modulated H3K9m2 levels. 3D model provides a relevant strategy to screen new drugs and SS is a promising drug against GBM that should now be tested in GBM animal models.