Yolande Richard

Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes

Differences in the composition of the hepatitis C virus (HCV) quasispecies between plasma and blood mononuclear cells (BMC) strongly suggest that BMCs support viral replication. We examined the frequency of such compartmentalization, the cell types involved, the constraints exerted on the different variants, and the role of immunoglobulin‐complexed variants. We screened the hypervariable region (HVR1) of HCV isolates from 14 HBsAg‐ and HIV‐seronegative patients with chronic HCV infection. HCV RNA was amplified and cloned from plasma, the immunoglobulin G (IgG)‐bound fraction, and total and sorted BMCs (CD19+, CD8+, CD4+, and CD14+ cells). Compartmentalization was estimated using a matrix correlation test. The ratio of nonsynonymous/synonymous substitutions (dN/dS ratio) was calculated for each compartment. HCV RNA was detected in 3/3 BMC, 11/11 CD19+, 10/11 CD14+, 4/11 CD8+ and 0/11 CD4+ cell samples. HVR1 sequences were significantly different between plasma and at least one cellular compartment in all nine cases analyzed, and between B cells (CD19+) and monocytes (CD14+) in all five available cases. IgG‐bound variants were distinct from cellular variants. DN/dS ratios were similar (n = 3) or lower (n = 6) in cellular compartments compared with plasma and the IgG‐bound fraction. In conclusion, HCV compartmentalization is a common phenomenon. B cells and monocytes harbor HCV variants showing a low rate of nonsynonymous mutations, a feature that might contribute to the persistence of HCV infection. (HEPATOLOGY 2004;39:817–825.)

Fractalkine receptor expression by T lymphocyte subpopulations and in vivo production of fractalkine in human

Expression and function of the fractalkine receptor CX3CR1 by T lymphocyte subpopulations was evaluated in healthy individuals. In CD8+ T lymphocytes, CX3CR1 was expressed by and functional in both CD45RO– and CD45RO+ cells. In CD4+ T lymphocytes, CX3CR1 was expressed mainly by CD45RO+ cells, and almost exclusively by activated HLA‐DR+ T lymphocytes. This receptor was functional in CD45RO+ cells, but not in CD45RO– cells. Expression of fractalkine was detected by in situ hybridization and immunohistochemistry in endothelial cells of normal lung and thymus. In hyperplastic lymph nodes, fractalkine was expressed by endothelial cells of high endothelial venules and of subcapsular vessels, by follicular dendritic cells (FDC) and by some follicle lymphocytes. Fractalkine mRNA was constitutively present in the HK FDC‐like cell line, and it was induced in vitro in B lymphocytes stimulated by an anti‐μ or by a CD40 mAb. These findings indicate that fractalkine may contribute to the recruitment of effector T helper lymphocytes, either in peripheral tissues or in lymphoid organs. In these tissues, fractalkine and its receptor may favor contact within follicles between activated T helper lymphocytes, activated B lymphocytes and FDC, thus contributing to the maturation of the B lymphocyte response.

IL4 and IL13 receptors share the γc chain and activate STAT6, STAT3 and STAT5 proteins in normal human B cells

IL13 induces the same biological effects as IL4 in normal human B cells. We show that as in the IL4R complex, both IL4Rα and IL2Rγc are components of the IL13R and that both cytokines induced STAT6, STAT3 and STAT5 activation in B cells. In spite of this similar downstream signalling, IL4 and IL13 used a different set of Janus kinases: IL13 is unable to activate JAK1 and JAK3.

Expression of Interleukin 13 Receptor in Glioma and Renal Cell Carcinoma: IL13Rα2 as a Decoy Receptor for IL13

Glioma and renal cell carcinoma (RCC) cells express high affinity interleukin 13 (IL13) binding sites, but only RCC cell proliferation was inhibited by IL13. Both of these two cell types are IL2-receptor γc chain-negative. We thus used these cell models to investigate the patterns of expression of IL13Rα1, IL13Rα2, and IL4Rα chains and the role of IL13Rα2 in the response to IL13. Using new specific antibodies and flow cytometry, we observed a similar surface expression of IL4Rα and IL13Rα1 chains in most RCC and glioma cells, whereas IL13Rα2 was only present on five of six glioma cell lines. In all glioma cell lines, the amount of IL13Rα2 expression was 10 to 30 times higher than that of the two other chains. Although there was no surface or intracellular expression of IL13Rα2, its mRNA was detected in three of seven RCC cell lines. The expression on RCC cells of IL13Rα2 mRNA and/or that of high-affinity IL13 binding sites is not sufficient to predict IL13Rα2 protein expression. Blocking experiments showed that IL4 and IL13 strongly inhibited RCC cell proliferation through a unique receptor composed of IL4Rα and IL13Rα1 chains. Using RCC cells stably transfected with IL13Rα2 cDNA, we showed that the overexpression of IL13Rα2 decreased the response to IL13 but not that to IL4. Our results demonstrate that IL13Rα2 acts as a decoy receptor for IL13 and that it may exert a tight regulation of IL13 activity without impairing the IL4 response of the same cell target.

Pivotal role of M-DC8⁺ monocytes from viremic HIV-infected patients in TNFα overproduction in response to microbial products.

HIV infects activated CD4⁺ T cells and induces their depletion. Progressive HIV infection leading to AIDS is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFα. This has been related to intestinal epithelial damage and microbial LPS translocation into the circulation. Using 11-color flow cytometry, cell sorting, and cell culture, we investigated the numbers and TNFα production of fully defined circulating dendritic cell and monocyte populations during HIV-1 infection. In 15 viremic, untreated patients, compared with 8 treated, virologically suppressed patients or to 13 healthy blood donors, circulating CD141 (BDCA-3)⁺ and CD1c (BDCA-1)⁺ dendritic cell counts were reduced. Conversely, CD14⁺ CD16⁺⁺ monocyte counts were increased, particularly those expressing M-DC8, while classical CD14⁺⁺ CD16⁻ M-DC8⁻ monocyte numbers were unchanged. Blood mononuclear cells from viremic patients produced more TNFα in response to LPS than those from virologically suppressed patients. M-DC8⁺ monocytes were mostly responsible for this overproduction. Moreover, M-DC8⁺ monocytes differentiated in vitro from classical monocytes using M-CSF and GM-CSF, which is increased in viremic patients plasma. This M-DC8⁺ monocyte population, which is involved in the pathogenesis of chronic inflammatory diseases like Crohn disease, might thus be considered as a major actor in the immune hyperactivation fueling HIV infection progression.

Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond

The immunological roles of B-cells are being revealed as increasingly complex by functions that are largely beyond their commitment to differentiate into plasma cells and produce antibodies, the key molecular protagonists of innate immunity, and also by their compartmentalisation, a more recently acknowledged property of this immune cell category. For decades, B-cells have been recognised by their expression of an immunoglobulin that serves the function of an antigen receptor, which mediates intracellular signalling assisted by companion molecules. As such, B-cells were considered simple in their functioning compared to the other major type of immune cell, the T-lymphocytes, which comprise conventional T-lymphocyte subsets with seminal roles in homeostasis and pathology, and non-conventional T-lymphocyte subsets for which increasing knowledge is accumulating. Since the discovery that the B-cell family included two distinct categories — the non-conventional, or extrafollicular, B1 cells, that have mainly been characterised in the mouse; and the conventional, or lymph node type, B2 cells — plus the detailed description of the main B-cell regulator, FcγRIIb, and the function of CD40+ antigen presenting cells as committed/memory B-cells, progress in B-cell physiology has been slower than in other areas of immunology. Cellular and molecular tools have enabled the revival of innate immunity by allowing almost all aspects of cellular immunology to be re-visited. As such, B-cells were found to express “Pathogen Recognition Receptors” such as TLRs, and use them in concert with B-cell signalling during innate and adaptive immunity. An era of B-cell phenotypic and functional analysis thus began that encompassed the study of B-cell microanatomy principally in the lymph nodes, spleen and mucosae. The novel discovery of the differential localisation of B-cells with distinct phenotypes and functions revealed the compartmentalisation of B-cells. This review thus aims to describe novel findings regarding the B-cell compartments found in the mouse as a model organism, and in human physiology and pathology. It must be emphasised that some differences are noticeable between the mouse and human systems, thus increasing the complexity of B-cell compartmentalisation. Special attention will be given to the (lymph node and spleen) marginal zones, which represent major crossroads for B-cell types and functions and a challenge for understanding better the role of B-cell specificities in innate and adaptive immunology.

Abnormal production of the TNF-homologue APRIL increases the proliferation of human malignant glioblastoma cell lines via a specific receptor

A proliferation-inducing ligand (APRIL) of the tumour necrosis factor (TNF) family is produced in small amounts in many tissues and more abundantly in tumours. APRIL has been reported to promote cell growth in vivo and in vitro. It was recently shown that the production of APRIL in some glioblastoma cell lines does not lead to an increase in cell growth. In this study, we investigated the production of APRIL and its ability to increase the proliferation of eight human glioblastoma cell lines. We found that APRIL was produced in the eight human glioblastoma cell lines tested but not in the normal embryonic astrocyte counterparts of glioblastomas. Flow cytometry demonstrated the presence of a specific APRIL-binding receptor on the cell surface in all the glioblastoma cell lines tested. This receptor was also present on normal embryonic and adult astrocytes and embryonic neural progenitor cells. Moreover, the addition of recombinant human APRIL resulted in an increase in proliferation rate of normal adult astrocytes and in four of eight cell lines tested. Addition of the soluble recombinant TNF-receptor-homologue B-cell maturation (BCMA) chimeric protein, which binds APRIL, confirmed the involvement of APRIL in the growth of malignant glioblastoma cell lines.

The Signaling Role of CD40 Ligand in Platelet Biology and in Platelet Component Transfusion

The CD40 ligand (CD40L) is a transmembrane molecule of crucial interest in cell signaling in innate and adaptive immunity. It is expressed by a variety of cells, but mainly by activated T-lymphocytes and platelets. CD40L may be cleaved into a soluble form (sCD40L) that has a cytokine-like activity. Both forms bind to several receptors, including CD40. This interaction is necessary for the antigen specific immune response. Furthermore, CD40L and sCD40L are involved in inflammation and a panoply of immune related and vascular pathologies. Soluble CD40L is primarily produced by platelets after activation, degranulation and cleavage, which may present a problem for transfusion. Soluble CD40L is involved in adverse transfusion events including transfusion related acute lung injury (TRALI). Although platelet storage designed for transfusion occurs in sterile conditions, platelets are activated and release sCD40L without known agonists. Recently, proteomic studies identified signaling pathways activated in platelet concentrates. Soluble CD40L is a good candidate for platelet activation in an auto-amplification loop. In this review, we describe the immunomodulatory role of CD40L in physiological and pathological conditions. We will focus on the main signaling pathways activated by CD40L after binding to its different receptors.

HIV Type 1 Glycoprotein 120 Inhibits Human B Cell Chemotaxis to CXC Chemokine Ligand (CXCL) 12, CC Chemokine Ligand (CCL)20, and CCL21

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40–50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or VH3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase Cβ3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, Gαi, and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials.

Lead Reduces Messenger RNA and Protein Levels of Cytochrome P450 Aromatase and Estrogen Receptor β in Human Ovarian Granulosa Cells

Abstract Exposure to lead causes decreased fertility in women. In the present study, we examined the in vitro effects of lead on cytochrome P450 aromatase (P450 arom) and on estrogen receptor β (ERβ), two key proteins for the human ovary. Aromatase is required for the bioconversion of androgen to estradiol; ERβ mediates estrogen effects in granulosa cells. Granulosa cells were collected from women undergoing in vitro fertilization and then cultured with 10 µM lead acetate. Using atomic absorption spectrometry, we showed that lead accumulated in cells. Aromatase activity as measured by a tritiated water production assay was significantly reduced. Using semiquantitative reverse transcription-polymerase chain reaction and Western blotting procedures, we showed that P450 arom and ERβ mRNA and protein content were both significantly reduced. Adding 10 µg/ml of cycloheximide, a protein inhibitor, did not eliminate the effects of lead. The present results support the hypothesis that the action of lead on fertility in women may result, in part, from the down-regulation of P450 arom and ERβ gene transcription in ovarian granulosa.