CD40-activated B cells from patients with systemic lupus erythematosus can be modulated by therapeutic immunoglobulins in vitro.

INTRODUCTION: Aberrant signaling within and between B and T cells, considered to be central in systemic lupus erythematosus (SLE), could depend on enhanced CD40-CD154 activation. As a result, autoreactive B cells, normally anergic, differentiate and secrete antibodies attacking several normal tissues. Thus restorating B cell homeostasis might help control this disease. In this study, two facets of SLE B cells were investigated, namely their in vitro response to CD40-CD154 and the effect of treatment with human immunoglobulins for intravenous use (IVIg). MATERIALS AND METHODS: Blood samples from SLE patients and healthy volunteers were obtained and used to isolate B cells, which were activated through CD40 in the presence or absence of IVIg. The phenotype, proliferation, and differentiation of the SLE B cells were determined and compared with those of control B cells using flow cytometry and standard ELISA. RESULTS: In this model, CD40-activated SLE B cells, as control B cells, proliferated and differentiated and were characterized by the emergence of CD19(lo)CD38(++)CD138(+)CD27(++) cells. IVIg treatment of the CD40-activated SLE B cells resulted in higher differentiation, characterized by increased secretion rates of IgG and IgM, as reported previously for control B cells. CONCLUSIONS: Taken as a whole, such accelerated differentiation of CD40-activated B cells suggests that IVIg may participate in re-equilibration of the antibody repertoire by replacing pathological antibodies by de novo harmless antibodies.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Whole-blood leukoreduction filters are a source for cryopreserved cells for phenotypic and functional investigations on peripheral blood lymphocytes.

BACKGROUND: Leukoreduction of blood is now widely performed by blood banks, and the possibility of recovering 10(8) to 10(9) white blood cells (WBCs) from leukoreduction filters, which are usually discarded, represents a promising source for normal human cells. Previous studies with these filters to prepare WBCs have performed their experimentation with fresh cells only. Whether these filter-derived cells could also be used to prepare frozen cell banks to facilitate work organization and open new avenues for their utilization as references in physiological studies and clinical investigations was investigated. STUDY DESIGN AND METHODS: Blood samples or whole-blood leukoreduction filters were obtained, after informed consent, from volunteers or blood donors, respectively. The proportions of CD3+, CD14+, CD16+, CD19+, and CD45+ cells within peripheral blood mononuclear cells (PBMNCs) were determined by flow cytometry from all samples. B cells were isolated and their functional responses were evaluated in vitro. RESULTS: The yield of PBMNCs recovered from whole-blood leukoreduction filters was lower (50%) than the one with fresh blood samples but still provided 2 x 10(8) to 4 x 10(8) PBMNCs per unit. After one cycle of freezing-thawing, the proportions of B- and T-cell populations were similar to normal blood values. Purified B cells issued from whole-blood leukoreduction filters displayed normal phenotypes and functions. CONCLUSION: Leukoreduction filters represent a valuable source of PBMNCs. These cells could be easily recovered to prepare frozen cell banks useful in basic phenotypic and functional analyses involving the main subsets of B cells and the global T-cell population.

B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase.

Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.

Differential responses of human B-lymphocyte subpopulations to graded levels of CD40-CD154 interaction.

Naïve and memory B-lymphocyte populations are activated by CD154 interaction through cell-surface CD40. This interaction plays an important role in the regulation of the humoral immune response, and increasing evidence indicates that fine variation in CD40 binding influences B lymphocytes, macrophages and dendritic cells in murine models. Here we have investigated whether and how variations in the intensity of the CD40-CD154 interaction could contribute to differential regulation of human B-lymphocyte populations. Proliferation and differentiation of B lymphocytes were monitored in response to graded levels of CD40 stimulation in the presence of interleukin (IL)-2, IL-4 and IL-10. Our results show that the level of CD154 binding to CD40 on B lymphocytes can directly influence the evolution of CD19(+) CD27(-) and CD19(+) CD27(+) cell populations. Furthermore, proliferation, global expansion of CD19(+) cells and emergence of CD38(++) CD138(+) cells, as well as immunoglobulin G (IgG) and IgM secretion, were affected by the level of exposure of B lymphocytes to CD154. These results suggest that the CD40-CD154 interaction is more like a rheostat than an on/off switch, and its variation of intensity may play a role in the regulation of B-lymphocyte activation following the primary and/or secondary humoral immune response.

Intravenous immunoglobulins induce the in vitro differentiation of human B lymphocytes and the secretion of IgG.

The therapeutic effects of intravenous immunoglobulins (IVIGs) in several autoimmune diseases are characterized by a decrease in pathologic autoantibody levels. Although little direct evidence has been reported in humans, the large repertoire of natural immunoglobulin G (IgG) antibodies in IVIGs is expected to be involved in the regulation of autoreactive B lymphocytes. In normal adult mice, IVIGs have been reported to modulate immature B cells as well as peripheral B lymphocytes through V-region connections. Studies with human serum also indicated that anti-idiotypic antibodies, present in IVIG preparations, could recognize both natural and pathologic autoantibodies. We have used an in vitro culture system to characterize the direct effect of IVIGs on human B lymphocytes. This in vitro culture system involves CD40 activation of B lymphocytes by its ligand CD154 in the presence of cytokines. In this system, addition of IVIGs decreased by 50% to 80% the expansion of B lymphocytes. This reduced expansion was due to a decrease in the proliferation rate. In addition, a portion of B lymphocytes was differentiated into IgG-secreting cells in the presence of IVIGs and the secreted IgGs were reactive with antigens such as nucleoprotamine, dsDNA, tetanus toxin, and human IgG F(ab')(2) fragments. These observations indicate that IVIGs can have direct effects on B lymphocytes and suggest that such IVIG regulation of B lymphocytes could be involved in the therapeutic effects of IVIGs in autoimmune diseases.