Prevention of T cell activation by interference of internalized intravenous immunoglobulin (IVIg) with MHC II-dependent native antigen presentation.

Activation of self-reactive CD4(+) T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4(+) T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4(+) T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4(+) T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4(+) T cell activation and the subsequent inflammatory response induced by these cells.

Inhibition of B cell-mediated antigen presentation by intravenous immunoglobulins (IVIg).

Previous work from our laboratory revealed that IVIg interacted with intracellular proteins involved in antigen presentation in B cells, suggesting that IVIg might interfere with the process of antigen presentation in these cells. In the present work, we used an in vitro assay with ovalbumin as model antigen and showed that IVIg inhibited both BCR-dependent and BCR-independent antigen presentation. The inhibition could not be explained by a modulation of expression of MHC II molecules expressed on B cells and was shown to occur in an FcgammaRIIb-independent manner, suggesting that the events responsible for the inhibitory effect occur at the intracellular level. This was supported by the observation of a direct correlation between the level of spontaneous internalization of two different proteins (IVIg and HSA) and their inhibitory potential. The inhibition of B cell-mediated antigen presentation reported here may help explain some of the anti-inflammatory effects of IVIg observed in treated patients, such as a decrease in autoantibody production.

Indirect inhibition of in vivo and in vitro T-cell responses by intravenous immunoglobulins due to impaired antigen presentation.

Several clinical studies done with intravenous immunoglobulin (IVIg)-treated autoimmune patients as well as several in vitro studies have revealed that IVIg can reduce polyclonal T-cell activation and modify their cytokine secretion pattern. However, their effect on (auto)antigen-specific T-cell responses has never been addressed directly. In the present work, we used an in vivo model of induction of antigen-specific T-cell responses and an in vitro antigen presentation system to study the effects of IVIg on T-cell responses. The results obtained showed that IVIg inhibited both the in vivo and in vitro antigen-specific T-cell responses but that this effect was the indirect consequence of a reduction in the antigen presentation ability of antigen-presenting cells. The inhibitory effect of IVIg was FcgammaRIIb-independent, suggesting that IVIg must interfere with activating FcgammaRs expressed on antigen-presenting cells to reduce their ability to present antigens. Such inhibition of T-cell responses by reducing antigen presentation may therefore contribute to the well-known anti-inflammatory effects of IVIg in autoimmune diseases.

Activation of cryptic IgG reactive with BAFF, amyloid beta peptide and GM-CSF during the industrial fractionation of human plasma into therapeutic intravenous immunoglobulins.

The mechanisms of therapeutic action of IVIg are still unclear in most autoimmune and inflammatory diseases. IVIg have been shown to bind to a variety of human proteins including BAFF, amyloid beta peptide and GM-CSF. It has been suggested that this autoreactivity could contribute to the therapeutic immunomodulatory effects of IVIg. In this work, we showed that native IgG purified from plasma under non-denaturing conditions were much less autoreactive than IVIg. However the native IgG autoreactivity with BAFF, amyloid beta peptide and GM-CSF was significantly increased by short incubation under the slightly denaturing conditions used during industrial plasma fractionation. We conclude that the relatively mild conditions used in industrial plasma fractionation are sufficiently denaturing to activate a significant amount of cryptic autoreactive plasma IgG which could be involved not only in the therapeutic immunomodulatory effects of IVIg but also in the adverse "allergic" reactions often observed in IVIg-infused patients.

Dose-dependent inhibition of BrdU detection in the cell proliferation ELISA by culture medium proteins.

Determination of the proliferation rate of cultured mammalian cells is widely done using incorporation of 5-bromo-2-deoxyuridine into replicating DNA followed by quantitative detection in ELISA using a specific monoclonal antibody. However, we noted that the BrdU ELISA results did not correlate with viable cell counts when increasing concentrations of proteins were added to test their effects on proliferating cells. This observation suggested that proteins could interfere with BrdU incorporation or detection in the commercial BrdU ELISA used. We show here that the presence of exogenous proteins during cell fixation and DNA denaturation significantly inhibited BrdU detection presumably by coating the extracted DNA by a concentration-dependent protein film. A simple modification to the manufacturer's protocol (cell washing) permitted to avoid this interference and resulted in a significant increase of the assay sensitivity.

Spontaneous internalization of IVIg in activated B cells.

Previous work from our laboratory showed that IVIg could directly influence the fate of human B cells by inducing their differentiation. The initial goal of the present study was to identify the cell surface molecules recognized by IVIg on human B cells. Purified resting and CD40-activated human B cells were incubated with IVIg and lysed prior to immunoprecipitation. The immunoprecipitated proteins were identified by mass spectrometry (LC-MS). This analysis revealed that BCR, as well as other cell surface receptors or membrane associated proteins were the main targets of IVIg. Surprisingly, intracellular proteins were also found in the immunoprecipitates, suggesting that IVIg could penetrate inside living cells and interact with intracellular targets. We have further studied this unexpected phenomenon and obtained evidence indicating that a significant amount of IVIg was spontaneously internalized inside living cells. We showed that IVIg internalization could occur in a BCR- and FcgammaR-independent pathway. Furthermore, spontaneous IVIg internalization was also observed in whole blood incubated with therapeutic concentrations of IVIg, even in presence of the high endogenous IgG concentration. These observations first suggest that spontaneous internalization can occur in IVIg-treated patients and also that some of the observed alterations in the physiology of IVIg-treated cells may not be only dependent on extracellular interactions of IVIg with cell surface receptors or soluble plasma proteins but may also involve intracellular interactions.

Increased secretion of hyperimmune antibodies following lipopolysaccharide stimulation of CD40-activated human B cells in vitro.

Human B cells can be cultured ex vivo for a few weeks, following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin-4 (IL-4). However, attempts to produce polyclonal antigen-specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen-specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells, recent data indicating that IL-4-activated human B cells are induced to express Toll-like receptor-4, the main LPS receptor, prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40-activated human B cells, accompanied by an increase in antigen-specific antibody secretion. This result suggested that some, but not all, B cells were able to differentiate into antibody-secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS-stimulated human B cells were induced to secrete higher amounts of IL-6, a pleiotropic cytokine well-known for its B-cell differentiation activity. In vivo, the effect of LPS on cytokine secretion by B cells may not only enhance B-cell differentiation but also help to sustain a local ongoing immune response to invading Gram-negative bacteria, until all pathogens have been cleared from the organism.

Characterization of the effects and potential mechanisms leading to increased megakaryocytic differentiation under mild hyperthermia.

The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently, we have demonstrated that CD34+ cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e., 39 degrees C). In this study, we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation, and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39 degrees C appear rapidly within 7 days, and that early transient culture at 39 degrees C led to even greater Mk yields (p<0.03). Surprisingly, cell viability was only found to be significantly reduced in the early stages of culture, suggesting that CB cells are able with time to acclimatize themselves to 39 degrees C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks, it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p=0.01). Conversely, gene arrays analysis demonstrated that Mks derived at 39 degrees C have a normal gene expression program consistent with an advanced maturation state. Finally, two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors.

Picogram doses of lipopolysaccharide exacerbate antibody-mediated thrombocytopenia and reduce the therapeutic efficacy of intravenous immunoglobulin in mice.

Exacerbation of antibody-mediated thrombocytopenia following infection with viruses has recently been demonstrated in a mouse model of the disease. The phenomenon was caused by an increased activation of phagocytes through gamma-interferon secretion in response to infection. Endotoxins from Gram-negative bacteria are also known to be potent activators of phagocytic cells. The objective of the present work was to determine whether lipopolysaccharide (LPS) could exacerbate antibody-mediated thrombocytopenia in vivo and so alter the therapeutic efficacy of intravenous immunoglobulin (IVIg), using a mouse model of thrombocytopenia. Very low doses of LPS (picogram range) and of anti-platelet antibodies (nanogram range), which did not induce thrombocytopenia individually, could synergize in vivo, resulting in significant decreases in platelet counts. The therapeutic efficacy of IVIg in antibody-mediated thrombocytopenia was significantly reduced in presence of LPS. These in vivo observations further support a role for bacterial infections in the aetiology of immune thrombocytopenic purpura (ITP) and may contribute to better understand the recognized lack of efficacy of IVIg in a significant proportion of patients with ITP.

Absence of cytokine modulation following therapeutic infusion of intravenous immunoglobulin or anti-red blood cell antibodies in a mouse model of immune thrombocytopenic purpura.

Human intravenous immunoglobulin (IVIg) and anti-D immunoglobulin preparations are used in the treatment of immune thrombocytopenic purpura (ITP). One mechanism proposed to explain their therapeutic effects in ITP patients is the induction of expression of anti-inflammatory cytokines, such as interleukin (IL)-10 or IL-1ra, leading to a reduction of phagocytic activity of the reticuloendothelial system. However, increased expression of pro-inflammatory cytokines was also noted following treatment of ITP patients, raising doubt on the actual contribution of anti-inflammatory cytokines in the therapeutic effects of IVIg and anti-D immunoglobulins. The present study evaluated the in vivo modulation of expression of a large array of inflammatory cytokines using a mouse model of thrombocytopenia. IVIg was not found to modulate cytokine expression although it efficiently prevented thrombocytopenia. In contrast, protective (M1/69) and non-protective (TER-119) anti-mouse red blood cell (RBC) antibodies (mimicking anti-D treatment) both increased the expression of CXCL-1 and CXCL-5. Thus, there was no relationship between inflammatory cytokine expression and prevention of thrombocytopenia by IVIg or anti-mouse RBC in the ITP mouse model. These results suggest that the increase in cytokine expression observed in ITP patients following IVIg or anti-D infusion is not required for their therapeutic effects but may rather represent a side-effect of the treatment.

Generation of kinins during preparation and storage of whole blood-derived platelet concentrates.

BACKGROUND: Leukoreduction of platelet (PLT) concentrates (PCs) may be associated with hypotension in recipients, and a role for bradykinin (BK)-related peptides has been proposed for this side effect. STUDY DESIGN AND METHODS: The concentration of BK and one of its vasoactive metabolites, des-arginine(9)-BK (des-Arg(9)-BK), was measured in a large number of PCs as a function of leukoreduction and storage duration with specific enzyme immunoassays and complementary techniques. RESULTS: On Day 0 of storage, kinins were detected in leukoreduced and unfiltered PCs at a concentration lower than 100 pg per mL. During storage, both kinin levels peaked on Day 5 of storage, with a concentration higher than 1 ng per mL in 22 percent of PCs whether filtered on Day 0 or not. Physicochemical and pharmacologic characterizations of immunoreactive kinins confirm their nature. In vitro activation of the contact system of the corresponding PLT-poor plasma showed that a high kinin concentration on Day 5 of the storage corresponded with a low kinin-forming capacity of plasma. On Day 7, BK was no longer elevated presumably due to its degradation and the depletion of kinin-forming capacity of the plasma in stored PCs. The activities of metallopeptidases that metabolize BK-related peptides in plasma from PCs were at levels similar to those recorded in the plasma of a normal reference population and were unaffected by storage. CONCLUSION: Storage of PCs contributes to the hydrolysis of high-molecular-weight kininogen and generation of pharmacologically relevant BK levels that might pose a hazard in susceptible patients.

Characterization of blood components prepared from whole-blood donations after a 24-hour hold with the platelet-rich plasma method.

BACKGROUND: The preparation of platelet (PLT) concentrates (PCs) from PLT-rich plasma (PRP) requires that whole blood (WB) be processed within 8 hours of collection. Increasing WB storage time to 24 hours would be logistically attractive. This study compares the in vitro quality of blood components prepared from WB stored for 8 and 24 hours at room temperature before processing with the PRP method. STUDY DESIGN AND METHODS: WB units were collected from ABO-matched blood donors. To reduce individual variations, paired donations were drawn in parallel, pooled, and split back in the collection bag. One unit was held for 6 to 8 hours and the other for 22 to 24 hours at 20 to 24 degrees C. Prestorage leukoreduced components were prepared with the PRP as intermediate product and analyzed during storage. RESULTS: RBC units prepared after an 8- or 24-hour hold were comparable in terms of hemolysis, sodium, pH, and ATP levels. RBC 2,3- diphosphoglycerate (2,3-DPG) was significantly lower in RBCs prepared from 24-hour hold donations immediately after processing but not after 20 days of storage. Residual white blood cells were approximately fivefold higher (p < 0.05) in 24-hour RBC units. For PCs, measurements for glucose, ATP, lactate, pH, extent of shape change, hypotonic shock response, and CD62p activation were similar. No differences were observed in the von Willebrand factor, factor (F)V, FVIII, and fibrinogen content of fresh-frozen plasma. CONCLUSIONS: The decrease in FVIII and RBC 2,3-DPG can be acceptable as a compromise to improve blood component logistics, but leukoreduction efficiency must be improved before considering the adoption of an overnight storage of WB before PRP processing.

Reversal of immune thrombocytopenia in mice by cross-linking human immunoglobulin G with a high-affinity monoclonal antibody.

Intravenous immunoglobulins (IVIgs) are used to treat an increasing number of autoimmune diseases, but their exact mechanism of action remains unknown. This study showed that cross-linking of human IgG present in IVIg preparations using a mouse monoclonal anti-human IgG generated complexes that prevented or reversed thrombocytopenia in mice more efficiently than IVIg. Furthermore, biologically active complexes were obtained simply by adding the monoclonal antibody to human serum. These results suggest the possible development of an IVIg-free substitute through the ex vivo, and possibly in vivo, formation of immune complexes containing autologous IgG of immune thrombocytopenic purpura patients.

High-throughput molecular profiling of blood donors for minor red blood cell and platelet antigens.

BACKGROUND: ABO and D phenotyping of both blood donors and patients receiving transfusions is routinely performed by blood banks to ensure compatibility. These analyses are performed by antibody-based agglutination assays. Blood is not tested for minor blood group antigens on a regular basis, however, because of cost and time constraints. This can result in alloimmunization of the patient against one to several minor antigens and may complicate future transfusions. STUDY DESIGN AND METHODS: To address this problem, an assay has been generated on the GenomeLab SNPstream genotyping system to test simultaneously polymorphisms linked to 22 different blood antigens with donor's DNA isolated from minute amounts of white blood cells. RESULTS: The results showed that both the error rate of the assay, as measured by the strand concordance rate, and the no-call rate were very low (0.1%). The concordance rate with the actual red blood cell (RBC) and platelet (PLT) serology data varied from 97 to 100 percent. Experimental or database errors as well as rare polymorphisms contributing to antigen conformation could explain the observed differences. These rates, however, are well above requirements because phenotyping and cross-matching will always be performed before transfusion. CONCLUSION: Molecular profiling of blood donors for minor RBC and PLT antigens will give blood banks instant access to many different matched donors through the setup of a centralized data storage system.

Autoantibody-induced formation of immune complexes in normal human serum.

Intravenous immunoglobulins (IVIg) are concentrated preparations of purified human plasma-derived IgG routinely used in the treatment of many autoimmune diseases. Their precise mechanisms of therapeutic action have remained unclear in most diseases and are attracting much interest due to the rapidly increasing use of this precious plasma-derived product. The presence in IVIg of IgG reactive with various human structures has been known for many years. In this review, we first briefly discuss the formation and role of natural autoreactive human IgG in healthy individuals. A role for IgG autoantibodies in the in vivo immunomodulatory effects of IVIg has been proposed several years ago but the underlying mechanism has remained unclear. Recent work has shown that the large IVIg doses infused in many patients could oversaturate the normal anti-idiotype-dependent inhibition of autoreactive IgG present in the plasma of all healthy individuals since the formation of autoimmune complexes could be observed in normal serum in presence of therapeutic amounts of IVIg. These autoimmune complexes could have potent in vivo immunomodulatory effects by interacting with various IgG and complement receptors. Furthermore the autoreactive IgG can be easily purified from IVIg by affinity chromatography, raising the interesting possibility of further fractionating IVIg into different sub-products for use in the treatment of different diseases. These results indicate that natural autoantibodies have important in vivo roles not only for the protection of the body against infectious agents but also for the efficiency of passive immunotherapy procedures used in the treatment of many diseases.

Efficient in vitro megakaryocyte maturation using cytokine cocktails optimized by statistical experimental design.

OBJECTIVE: A multi-step statistical strategy was applied to quantify individual and interactive effects of cytokines on megakaryopoiesis and to determine the concentration of the selected cytokines that optimize ex vivo megakaryocyte (MK) expansion, maturation, and platelet production in stromal- and serum-free conditions. MATERIALS AND METHODS: Immature MK were first generated from human CD34(+)-enriched cord blood cells cultured for 7 days in conditions favoring MK commitment. Then, the effect of different combinations of cytokines at various concentrations on MK differentiation and platelet production was tested on the day-7 MK. RESULTS: A large-scale screening of 13 cytokines in the presence of thrombopoietin (TPO) using Placket-Burman designs (PBD) was initially performed to identify stimulators of MK maturation. Afterwards, a statistical analysis of the two-level factorial designs revealed that in the presence of TPO, MK maturation was significantly stimulated by stem cell factor (SCF), interleukin (IL)-6, and IL-9, whereas Flt-3 ligand (FL) had a positive effect only on the expansion of MK progenitors. In contrast, erythropoietin (EPO) and IL-8 were inhibitors of MK maturation. A response surface methodology was then used to optimize the concentrations of the selected cytokines (TPO, SCF, IL-6, and IL-9) and defined a new cytokine cocktail that maximized MK expansion and maturation. Importantly, the increased MK output was accompanied by a very high MK purity ( approximately 90%). Another optimum was also found at a higher SCF concentration, which further improved MK expansion and maturation, but reduced MK purity. CONCLUSION: These statistical methods provide an efficient tool to analyze complex systems of cytokines and to develop promising ex vivo MK culture systems for clinical applications.

Therapeutic intravenous immunoglobulins.

Intravenous immunoglobulins (IVIg) are concentrated formulations of human IgG prepared by industrial fractionation of large pools of individual plasma donations. IVIg were developed 20 years ago for the prophylaxis support of immunodeficient patients. However, IVIg have been increasingly used since 10 years, in the treatment of many autoimmune and inflammatory diseases raising the possibility of product shortages and ever increasing costs in the near future. Surprisingly, the immunomodulatory mechanisms of action of IVIg are unclear because of the diversity and often contradictory Fc, F(ab')(2), and non-IgG-related mechanisms that have been proposed from clinical observations and from results obtained in various in vitro and in vivo experimental models. These concepts are reviewed here and we discuss in more details three areas of active research, namely the mechanisms of IVIg action in Idiopathic Thrombocytopenic Purpura (ITP), the effects of IVIg on activated B lymphocytes and the possible involvement of autoantibodies of IgG isotype (auto-IgG) in the immunomodulatory effects of IVIg. The elucidation of the mechanisms of action of IVIg is crucial for a more rationalized clinical use of IVIg and for developing substitutes for some of the immunomodulatory indications in order to ensure long-term availability of plasma-derived IVIg for immunodeficient patients.

The survival of IL-6-dependent myeloma cells critically relies on their capability to transit the G1 to S phase interval of the cell cycle.

Interleukin-6 (IL-6) has an essential role in the initial progression of myeloma cell tumours. IL-6 triggers proliferation of these cells via the Ras-mitogen-activated protein kinase (MAPK) cascade and is thought to promote their survival via signal transducer and activator of transcription (STAT) pathway-dependent regulation of Bcl-2 family antiapoptotic members. Using IL-6-dependent murine B9 hybridoma/plasmacytoma cells, we here report that exiting the cell cycle G1 phase is a crucial step contributing to maintain viability. We show that (1) drug-mediated reversible G1 arrest triggered apoptosis despite the presence of IL-6; (2) a short IL-6 pulse to G1-arrested cells was sufficient to induce S phase entry and prevent apoptosis; and (3) phorbol ester and related derivatives promoted S phase entry and survival of IL-6-starved cells without up-regulating bcl-XL expression. Furthermore, that the MAPK kinase (MEK) 1/2 inhibitor, U0126, blocked proliferation and induced death of B9 cells indicate that IL-6 may not exert its survival effect primarily through bcl-XL and emphasizes the key role of Ras-MAPK cascade elements in the regulation of myeloma growth/viability.

Iron specific growth inhibition of Burkitt's lymphoma cells in vitro, associated with a decrease in translocated c-myc expression.

The cellular proto-oncogene c-myc is an important transcription factor that plays a role in several cellular activities such as proliferation, differentiation, and apoptosis. It follows that regulation of c-myc expression is crucial for maintaining cell integrity. Amplification or translocation can convert this proto-oncogene into an activated oncogene, thereby deregulating c-myc expression. Changes in the expression of c-myc leading to malignant cell growth and tumor progression can also be triggered by extrinsic factors. It has been reported that iron can increase cell proliferation, mainly by stimulating DNA synthesis as well as by enhancing c-myc expression. Here, we studied the effect of iron on cells in which c-myc expression is deregulated by either chromosomal translocation or gene amplification. When added to Burkitt's lymphoma cell lines, iron markedly inhibits cell proliferation. This effect is mediated by a cell cycle arrest in G2/M, followed by an important decrease in c-myc expression, whereas no effect could be observed in a cell line harboring amplified c-myc. Moreover, the down-regulation of c-myc expression, which is independent from cell cycle blockade, leads to cell death by apoptosis. Collectively, our results suggest the existence of a novel iron-dependent cell cycle regulatory mechanism involving modulation of translocated c-myc gene expression.