Abstract: BACKGROUND: Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-kappaB (nuclear factor kappaB) and STAT (signal transducer and activator of transcription) families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-kappaB and STAT rely on specific ELISAs, Western Blots and--most recently described--flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. RESULTS: The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells). To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1beta, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA); the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-kappaB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. CONCLUSION: In response to IL1beta, or IL10, the levels of phosphorylated NF-kappaB and STAT3--respectively--increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages--but, interestingly, not T-lymphocytes (in the context of PBMCs)--responded significantly to sCD40L by increasing phosphorylated NF-kappaB.
Abstract: Platelets have long been confined to haemostasis only. However, novel functions for platelets have been identified recently. Those non-nucleated cells indeed participate to inflammation and also they produce and release numerous factors with known immunomodulatory functions. Among those factors are cytokines and chemokines and the like, such as soluble CD40-Ligand (CD154), which are key molecules in that they bridge innate and adaptative immunity; sCD40L is active on T cells, B cells, monocytes and macrophages, dendritic cells and endothelial cells lining the blood vessels. This means that when a platelet concentrate is transfused to a recipient, a huge amount of cytokines and chemokines is also infused. In this state of the art review, we will present arguments on the role of platelet secretory products in modulating cellular parameters of immunity, and--very likely--in altering functions of those immune cells upon encounters while infusing platelets in blood recipients. We aimed at summarizing data that have been made available on the issue of cytokines/chemokines released by stored platelets prior to delivery. We will focus on the suspected role of the CD40/CD40L tandem in postplatelet transfusion reactions or incidents. We will present recent data on the role of pathogen inactivators on the docking and/or release of cytokines/chemokines by platelets.
Abstract: OBJECTIVE: Blood platelets represent a link between hemostasis, inflammation, and tissue repair. Their role in immune responses and inflammation mainly involves many molecules, among which Toll-like receptor, major histocompatibility complex class I, CD40 and CD154/CD40 ligand (CD40L). As platelets are the major purveyor of soluble CD40L (sCD40L), we sought to determine their involvement in CD40/CD40L-dependent immune responses and to understand the interactions between platelets and peripheral B lymphocytes. MATERIALS AND METHODS: We examined the capacity of platelets to bind nonstimulated B cells, and phenotypic changes by flow cytometry and confocal scanning laser microscopy. Modulation of cytokines/chemokines and total levels of immunoglobulin (Ig) A, IgG, IgM, and IgG subclasses in supernatants of coculture, platelets, and B lymphocytes was performed by sandwich enzyme-linked immunosorbent assay and differential production of cytokine mRNA as determined by reverse transcriptase polymerase chain reaction. RESULTS: In coculture, platelets and B lymphocytes were mutually activated, as demonstrated by the increased expression of platelet CD62p and B-cell CD86. Platelet/B-cell interactions were accompanied by changes in membrane expression of CD40 and CD40L by both platelets and B lymphocytes. IL12p70 and IL8 gene transcription were significantly reduced, which was attributable to B cells. Conversely, there was a significant, platelet-dependent reduction of sCD40L and RANTES mRNA expression. After a 3-day incubation with platelets, differentiated B cells increased their in vitro production of IgG1, IgG2, and IgG3, but not IgG4, IgA, or IgM. CONCLUSION: These data emphasize the potentially important role of platelets in the adaptive immune response. Platelets have an immunoregulatory role that might be applied clinically in multitransfused patients (e.g., hematopoietic stem cell transplantation).
Abstract: BACKGROUND: Blood platelets (PLTs) link the processes of hemostasis and inflammation. Recent studies have demonstrated that PLTs promote immunity and inflammation mainly by means of the CD40/CD40L pathway. Our objective was to describe the accumulation of cytokines in PLT concentrates during storage. STUDY DESIGN AND METHODS: Pools of PLT concentrates were prepared, separated from plasma, and resuspended in clinical-grade storage medium; samples were taken on Days 0, 1, 2, 3, and 5 for analysis, without replacement (i.e., without soluble protein dilution). Interleukin (IL)-6, IL-8, PLT-derived growth factor (PDGF)-AA, soluble CD40 ligand (sCD40L), RANTES, and transforming growth factor-beta production were measured by specific enzyme-linked immunosorbent assays. RESULTS: Over time, the levels of RANTES, IL-8, and IL-6 were stable. In contrast, the levels of PDGF-AA and sCD40L increased. Ex vivo production of sCD40L was quantified at levels sufficient to induce B-cell effects based on previous studies of in vitro induced B-cell activation and differentiation by sCD40L. Cytokine and/or chemokine levels were generally higher in PLT concentrate supernatants and/or PLT lysates in comparison to PLT-free plasma, allowing the determination of which cytokine and/or chemokine was absorbed or secreted by transfusion-grade PLTs over time. CONCLUSION: Our data provide evidence that stored PLTs contain molecules with known immunomodulatory competence and secrete them differentially over time during storage for transfusion purposes.
Abstract: Platelets are primarily involved in thrombosis and haemostasis, and they have recently been shown to have a role in innate immunity and in inflammation. We have determined the markers of innate immunity that are expressed by platelets, specifically the Toll-like receptors (TLR), originating from mixes of platelet concentrates (MPC, n = 5) between day zero and day five after blood collection. The surface membrane and intracellular expression of TLR were measured, both after and without permeabilization, using flow cytometry. We observed weak expression of TLR2, TLR4 and TLR9 on the surface of CD41(+) platelets. The expression levels of TLR4 were high (59 +/- 2.2%). Moreover, there was a significant expression of TLR2 (47.5 +/- 4.8%), TLR4 (78.8 +/- 1.3%) and TLR9 (34.2 +/- 7.5%) in the cytoplasm of CD41(+) platelets. The expression of the three receptors did not change significantly during the course of the 5 day observation period. The percentage of TLR expression is significantly modulated between activated versus non-activated platelets, both after and without permeabilization (P < 0.01). Study of the expression of TLR could increase our knowledge of the level of platelet participation during an immune reaction and inflammation. In the same way as the platelet ligand/receptor pair CD40L/CD40 is, the TLR are expressed by platelets, and could serve as a link between innate and adaptive immunity.
Abstract: As B-lymphocytes play an important role in innate and adaptive immunity, we aimed to examine the effects of CpG oligodeoxynucleotides (ODNs) on purified tonsil-originating CD19+ B-cells, representing mucosal B-cells. We screened various K-type ODNs, reactive with human B-cells, and tested for the production of immunoglobulins in vitro. Using one CpG-ODN, DSP30, we observed that it could upregulate not only Toll-like receptor 9 (TLR9) mRNA expression in activated B-cells, but also the early expression of CD69 followed by the sequential expression of CD80, CD86 and the nuclear factor (NF)-kappaB pathway. Furthermore, mRNA expression of certain B-cell-derived cytokines was influenced by exposure to DSP30, with a strong upregulation of interleukin 6 (IL-6) and downregulation of IL1-beta. Stimulation of B-cells, co-stimulated with IL-2, IL-10 and soluble CD40 ligand (sCD40L) with different CpG-ODNs, had differing effects on the terminal differentiation in vitro of B-cells into immunoglobulin-secreting cells. TLR9 is involved in innate immunity and the recognition of bound CpG DNA from invading bacterial pathogens. As tonsillar B-cells are mucosal-type B-lymphocytes, this study suggests that CpG-ODNs show promise as mucosal adjuvants in modulating the local production of immunoglobulins of certain classes and subclasses, a crucial issue in vaccine perspectives.
Abstract: With the addition of various cytokines, the CD40-CD40 ligand (CD40L) system can act as a T-helper cell surrogate to permit B lymphocytes to produce large amounts of polyclonal Ig. In the present study, we tested six CD40-CD40L stimulation models: (i, ii) soluble agonistic 89 and G28.5 mAbs ; (iii, iv) 89 and G28.5 bound via their Fc fragments on CDw32-transfected mouse fibroblasts; (v) purified, soluble, trimeric human CD40L molecules (sCD40L); and (vi) human CD40L expressed by a CD40L-transfected mouse fibroblastic cell line (LCD40L). Target B cells consisted of purified blood and tonsillar CD19+ lymphocytes cultured in the presence of CD40 stimuli and IL-2 and IL-10, added at the onset of each B cell culture. A) There was differential expression of CD69, CD80 and CD86 exposure to sCD40L and LCD40L was ensued by the strongest % MFI changes over control. B) In blood B cells, mAbs and sCD40L induced IgA, IgM and IgG production almost equally well; LCD40L proved less efficient. In contrast, in tonsil B cells, LCD40L induced significantly more IgA, IgG1, IgG3 and IgM production than other signals. Using certain CD40/CD40L stimuli to model in vitro Ig production, a system used regularly in many laboratories, may affect the interpretation based on the cell type and on the CD40/CD40L system used.
Abstract: Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1beta through the nuclear factor-kappaB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.
Abstract: To better understand the pathophysiology of B cell populations-the precursors of antibody secreting cells-during chronic human immunodeficiency virus (HIV) infection, we examined the phenotype of circulating B cells in newly diagnosed Africans. We found that all African individuals displayed low levels of naive B cells and of memory-type CD27+ B cells, and high levels of differentiated B cells. On the other hand, HIV-infected African patients had a population of germinal center B cells (i.e. CD20+, sIgM-, sIgD+, CD77+, CD138(+/-)), which are generally restricted to lymph nodes and do not circulate unless the lymph node architecture is altered. The first observations could be linked to the tropical environment whereas the presence of germinal center B cells may be attributable to chronic exposure to HIV as it is not observed in HIV-negative African controls and HAART treated HIV-infected Europeans. It may impact the management of HIV infection in countries with limited access to HIV drugs and urges consideration for implementation of therapeutic vaccines.
Abstract: We studied isotype profiles of anti-HIV antibodies (Ab) in HIV-1-infected African patients with high viral loads and major B cell dysfunction. We focussed on IgG1, IgG3, and IgA as these classes and subclasses tend to support neutralizing functions against HIV. Total IgG1, IgG3 and IgA were detected in the plasma of both HIV-1-infected and HIV-negative African individuals, but there was significantly more IgG3, a rare subclass, in HIV-1-infected patients (P < 0.05). Anti-HIV-gp160 specific antibodies were detected in sera from nearly all HIV-1-infected individuals tested, but not in HIV- individuals: 10/10, 9/10 and 8/10 individuals displayed specific IgG1, IgG3 and IgA, respectively. In the corresponding PBMC cultures carried out in the presence of IL-10 and IL-2, there was specific IgG1 and IgA in 5/10, and 3/10, respectively, but no IgG3 was detected. When HAART-treated European HIV-infected PBMC cultures were tested using the same protocol, specific IgG3 was detected in 4/10 cultures, and was unaffected by the addition of soluble CD40L molecules. The present study thus shows that, despite lymph node disorganization in HIV-infected drug-naïve Africans, these individuals retain the ability to produce HIV-specific IgG1, IgG3 and IgA. However, the specific mechanisms controlling the selective production of IgG3, probably the most potent subclass and a potential target of immuno-intervention, warrants further investigation.
Abstract: Unseparated peripheral blood mononuclear cells (PBMCs) obtained from drug-naïve African individuals living in a context of multi-infections and presenting with high viral load (VL), were cultured in vitro and tested for their ability to produce antibodies (Abs) reacting with HIV-1 antigens. Within these PBMCs, circulating B cells were differentiated in vitro and produced IgG Abs against not only ENV, but also GAG and POL proteins. Under similar experimental conditions, HAART treated patients produced Abs to ENV proteins only. The in vitro antibody production by drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10) but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was not abrogated by the addition of various monoclonal Abs (mAbs) to co-stimulatory molecules. This suggests that the in vitro antibody production by drug-naïve individuals' PBMCs resulted from the maturation of already envelope and core antigen-primed, differentiated B cells, presumably pre-plasma cells, which are not known to circulate at homeostasy. As in vitro produced Abs retained the capacity of binding antigen and forming complexes, this study provides pre-clinical support for functional humoral responses despite major HIV- and other tropical pathogen-induced B cell perturbations.
Abstract: HIV1-gp160 holds promises in anti-HIV vaccinal strategies. However, this molecule has been described to exhibit superantigenic activities. The present study aimed at examining the effect(s) of HIV1-gp160 on human B cells and in particular on B cells originating from HIV- donors. We purified human B cells of various origins, i.e. from blood and from tonsils (representing a mucosal-type origin), and we tested these cells (stimulated with a polyclonal B cell activator, interleukin (IL)-2 and IL-10 as cytokines, and recombinant HIV1-gp160) for the production of IgG and IgA in an in vitro model. Gp160 induced significantly less total IgG by blood - but not tonsil-originating - B cells and did not affect total IgA production. Further, HIV1-gp160 up-regulated IL-2-, IL-4- and IL-10-mRNA levels in stimulated blood B cells (these cytokines are known to be active on B cell activation and differentiation). Interestingly, HIV1-gp160 also up-regulated IL-1beta-, transforming growth factor (TGF)-beta-, interferon (IFN)-gamma- and IL-12-mRNA levels in stimulated mucosal-type, tonsil-originating, B cells. As these latter cytokines are involved in proinflammatory activities, HIV-gp160 delivery at the mucosal sites would be compatible with an adjuvant activity.
Abstract: This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro, in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production, and possibly IgA, specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-beta led to the production of specific IgG3 or IgG1 and/or IgA, respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes.
Abstract: We aimed at examining NFkappaB translocation in B lymphocytes during in vitro activation through the specific receptor for antigen using a technique convenient in most laboratories such as flow cytometry. We present here an original, convenient, and reproducible technique to study B cell activation events through NFkappaB translocation by means of a novel, specific flow cytometry assay. Intranuclear translocation of NFkappaB p65 was induced after a 45min stimulation; the highest signal was detected for a 10 ng/ml stimulus compared to the unstimulated condition (P< 0.05). Purified CD19+ B cells--cultured in the presence of optimal concentrations of anti-micro fragment Abs (10ng/ml) for 45min at 37 degrees C--induced a mean 60% (range: 45-67%) MFI-- and thus, nuclear NFkappaB translocation-increase. We observed a one-pike profile of NFkappaB staining in PBMC B cells and a two-pike profile of NFkappaB staining in using tonsil B cells. B cells are susceptible to various dysregulations leading to minor to severe pathology (including immunoproliferative disorders). Studies of signal transduction carried out specifically in human B cells, using a novel technique gave considerable advantages: feasibility, sensitivity, reproducibility, ease.
