ABSTRACT
Intravenous immunoglobulin (IVIg) is a therapeutic preparation of plasma-derived human IgG and is increasingly used for the treatment of several neurological inflammatory disorders. However, it is not clear whether the IgG molecules contained in IVIg can actually cross the BBB in treated patients. We recently showed that LRP1, an endocytic receptor involved in transcytosis of several proteins across the BBB was able to interact with IVIg. In the present study, we show that LRP1 is involved in IVIg internalization inside living cells. Our data also suggest that following internalization, IVIg is recycled to the cell surface, raising the possibility that LRP1 can mediate IVIg transcytosis across the BBB. Finally, we show that IVIg-LRP1 interaction leads to LRP1 tyrosine phosphorylation.
Subject(s)
Blood-Brain Barrier/metabolism , Immunoglobulins, Intravenous/metabolism , Receptors, LDL/metabolism , Transcytosis , Tumor Suppressor Proteins/metabolism , Animals , Biological Transport/physiology , Cell Line , Low Density Lipoprotein Receptor-Related Protein-1 , Mice , Phosphorylation/physiology , Tyrosine/metabolismABSTRACT
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.
Subject(s)
Antigen Presentation/immunology , CD4-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class II/immunology , Immunoglobulins, Intravenous/immunology , Lymphocyte Activation/immunology , Animals , Cell Line , Dendritic Cells/immunology , Female , Immunoglobulins, Intravenous/pharmacology , Lymphocyte Activation/drug effects , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Ovalbumin/immunologyABSTRACT
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.
Subject(s)
Bromodeoxyuridine/metabolism , Cell Proliferation , Culture Media/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Proteins/metabolism , Cell Count/methods , Cell Proliferation/drug effects , Cells, Cultured , Humans , Jurkat Cells/metabolism , Proteins/administration & dosage , Sensitivity and SpecificityABSTRACT
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.