Differential activation of signal transducer and activator of transcription 6 in B cells from allergic children and their non-allergic siblings.

BACKGROUND: The germline (GL) epsilon promoter is regulated by IL-4 and is essential for class switching to IgE. IL-4-induced gene expression is largely mediated through activation of latent transcription factor STAT6 (signal transducer and activator of transcription 6). OBJECTIVE: We investigated whether increased levels of IgE in allergic individuals may be associated with alteration in the level or activation of STAT6 and subsequent increase in GL epsilon promoter activity. METHODS: Electrophoretic mobility shift assay and Western blotting assays were used to investigate the level of expression and activation of STAT6 in Epstein-Barr virus (EBV)-transformed B cell lines from children with birch pollen allergy and their non-allergic siblings. The activity of the GL epsilon promoter was tested in a transient transfection assay. RESULTS: STAT6 was expressed at the same level in all B cell lines tested. In two out of five sibling pairs STAT6 was activated by IL-4 more efficiently in the allergic individuals but in the three other pairs the opposite was found. In transient transfections, no difference in IL-4-induced GL epsilon promoter function was detected, although basal promoter activity varied between allergic and healthy siblings in two out of five pairs. CONCLUSIONS: We demonstrate for the first time that upon IL-4 signalling STAT6 transcription factor activation differs in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude a possibility that stronger activation of this transcription factor is associated with an expression of allergic phenotype.

Regulation of adhesion and motility in B lymphocytes.

During the differentiation process of B lymphocytes, they go through changes in adhesion and motility. In order to investigate the molecular mechanism of such changes, in vitro culture systems are necessary. When B cells are activated by various stimuli, they form different types of homotypic aggregates. In addition, they might also spread and express microvilli and/or become polarized, the latter being a sign of motility. In this review, we summarize our own research in this area. We give evidence for involvement of different adhesion and signalling molecules, and by the end, we speculate on the in vivo significance of our findings.

HS1,2 enhancer regulation of germline epsilon and gamma2b promoters in murine B lymphocytes: evidence for specific promoter-enhancer interactions.

During an immune response, activated B cells develop into high rate Ig-secreting plasma cells. They also switch from production of IgM to IgG, IgA, or IgE. This process requires a DNA recombination event, which is regulated at the transcriptional level by the production of isotype-specific, sterile germline (GL) transcripts. Induction of these transcripts is controlled by GL promoters and, possibly, by IgH 3' enhancers. We investigated the interaction of the GL epsilon and gamma2b promoters with the HS1,2 enhancer using transiently transfected mouse primary B cells and cell lines. The constructs used for the transfections contained a GL promoter upstream and HS1,2 downstream of a luciferase reporter gene. Both GL epsilon and gamma2b promoters synergized strongly with the HS1,2 enhancer in activated primary B cells, a mature B cell line, and a plasma cell line. We show that the major activity of HS1,2 in activated primary B cells occurs within a 310-bp fragment that includes NF-kappaB, OCT, and NF of activated B cells (Ets/AP-1) sites. By mutating the consensus sequences for various transcription factors, we have determined which sites in HS1,2 are important for synergy with the GL epsilon and gamma2b promoters. Our findings indicate that different sites in HS1,2 might selectively interact with the GL epsilon and gamma2b promoters. We also provide evidence that B cell-specific activator protein is not an absolute suppressor of HS1,2 activity.

Cdc42, Rac1, and the Wiskott-Aldrich syndrome protein are involved in the cytoskeletal regulation of B lymphocytes.

Patients with the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) have lymphocytes with aberrant microvilli, and their T cells, macrophages, and dendritic cells are impaired in cytoskeletal-dependent processes. WAS is caused by a defective or a missing WAS protein (WASP). Signal mediators interleukin-4 (IL-4) and CD40 are important for actin-dependent morphology changes in B cells. A possible function of WASP and its interacting partners, Cdc42 and Rac1, was investigated for these changes. It was found that active Cdc42 and Rac1 induced filopodia and lamellipodia, respectively, in activated B cells. Evidence is given that IL-4 has a specific role in the regulated cycling of Cdc42 because IL-4 partially and transiently depleted active Cdc42 from detergent extract of activated B cells. WASP-deficient B lymphocytes were impaired in IL-4-- and CD40-dependent induction of polarized and spread cells. Microvilli were expressed on WASP-deficient B cells, but they appeared shorter and less dense in cell contacts than in wild-type cells. In conclusion, evidence is provided for the involvement of Cdc42, Rac1, and WASP in the cytoskeletal regulation of B lymphocytes. Aberrations in WASP-deficient B lymphocytes, described here, provide further evidence that WAS is a cytoskeletal disease of hematopoietic cells. (Blood. 2001;98:1086-1094)

STAT6 is required for the regulation of IL-4-induced cytoskeletal events in B cells.

During lymphocyte activation, changes in cell morphology are commonly observed. This reflects cell functions important for the regulation of immune responses such as cell adhesion or cell migration. Notably, IL-4 has been shown to induce adhesion and locomotion in B cells, and we have recently described that IL-4 causes dramatic changes in B cell morphology. Thus, such B cells spread with dendritic cell protrusions and produce microvilli-like structures. The molecular mechanisms by which IL-4 induces these complex changes are currently unknown. Two signal transduction pathways are well described for IL-4, i.e. one involving insulin receptor substrate (IRS)-2 and a Janus kinase (JAK)/ signal transducer and activator of transcription (STAT) pathway mediated by STAT6. In this study we therefore used B cells from STAT6-deficient mice to address the question of a possible STAT6 dependence in IL-4-induced morphology changes. By light and electron microscopy, cell spreading and polarization were found to be severely impaired and microvilli formation was reduced. In contrast, only mild impairment was observed in cell adhesion in B cells from STAT6-deficient mice. Our results show that adhesion can be induced in the absence of STAT6. However, expression of STAT6 is necessary for optimal responses in both cell adhesion and microvilli induction. STAT6 is also essential to allow an IL-4-dependent spreading or polarization response. A possible interpretation of our results is that STAT6-dependent expression of a specific gene or genes is required for IL-4 to affect changes in B cell morphology.

Characterization of CD40-dependent immunoglobulin class switching.

Previous studies have shown that signalling via CD40 together with cytokines stimulates immunoglobulin (Ig) class switching in B cells. This process includes induction of germline (GL) transcripts and switch recombination. Using an agonistic rat anti-mouse CD40 monoclonal antibody (MoAb), we investigated the role of CD40 signalling in these molecular events. We found that stimulation of murine B cells induced high steady-state levels of germline gamma1, gamma2b and low levels of epsilon transcripts. No detectable gamma2a or alphaRNA were found and the level of gamma3 transcripts was high both in stimulated and unstimulated cells. Although cells treated with anti-CD40 MoAb had high levels of GL gamma1 and gamma3 transcripts, we failed to detect switching to IgG1 or IgG3. However, anti-CD40 MoAb-stimulated cells increased expression of IgG2b. Interestingly, anti-CD40 plus interleukin (IL)-5 induced switching to IgG1. Previous work has demonstrated that CD40 signalling, but not lipopolysaccharide (LPS), induces the gamma1 promoter and that NF-kappaB motifs are important. We show here that both LPS and anti-CD40 activated NF-kappaB proteins binding to the gamma1 promoter. The bound NF-kappaB complexes were different with regard to total concentration and subunit composition. In the light of our data, the mechanism of CD40-mediated Ig class switching is discussed.

Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes.

Lymphocyte activation is often accompanied by changes in cell morphology, for example, in cell adhesion or motility. IL-4 is a cytokine exerting many effects on B lymphocytes. In this study, we show that stimulation with LPS in combination with IL-4, but not LPS or IL-4 alone, results in a pronounced dendritic morphology of B cells. Using a culture system in which Abs directed to B cell surface markers are immobilized on the tissue culture plastic, we find that cell spreading can be mediated by a variety of Abs, including anti-CD44, -CD23, -LFA-1, -VLA-4, -ICAM-1, and -Ig. B cells stimulated with anti-Ig Abs plus IL-4, or anti-CD40 Abs in the presence or absence of IL-4, are also induced to spread, while IL-2, IL-5, or IL-10 in combination with LPS or alone fail to induce this. Spreading correlates with induction of tight cell aggregation. It is sensitive to cytochalasin B, indicating a requirement for intact actin cytoskeleton. CD44 is selectively detected in the detergent-insoluble fraction of cell lysates prepared from LPS plus IL-4-stimulated B cell cultures after Ab cross-linking of CD44, suggesting a membrane protein-cytoskeleton interaction. Interestingly, electron microscopy studies reveal induction of microvilli-like structures on LPS plus IL-4-stimulated blasts, suggesting that IL-4 can influence cell morphology on an ultra-structural level. In summary, our data show that stimulation with LPS plus IL-4 or ligation of CD40 is capable of inducing dramatic morphologic changes in murine B cells, which correlates with in vitro induction of strong cell adhesion.

Assessment of the role of leucocyte function-associated antigen-1 in homotypic adhesion of activated B lymphocytes.

In this study we investigated how T-cell-dependent stimuli, via interleukin-4 (IL-4) or CD40 ligation, influence homotypic B-cell adhesion when compared with induction by the T-cell-independent stimulus lipopolysaccharide (LPS). Using primary murine B cells, we found that T-cell-dependent stimulation led to increased aggregation as compared to that induced by LPS. The adhesion was to a large extent dependent on the adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1). We found that activation of B cells with the mitogenic stimuli induced an increased avidity of LFA-1 for its ligand, intercellular adhesion molecule-1 (ICAM-1). The increase was stable and different from that induced by phorbol esters. Although adhesion was reduced using B cells from LFA-1(-/-) mice, aggregation occurred in response to T-cell-dependent stimuli. Our data suggest that adhesion of B lymphocytes is regulated in different modes. One is induced by antigen and leads to a transient conformational change of the LFA-1 molecule. Another is induced by mitogenic stimuli and leads to stable avidity increase of LFA-1, possibly via activation of cytoskeletal anchorage. A third is LFA-1 independent, of low avidity and is induced by T-cell-dependent stimuli.

Allotype-associated variation in the human gamma3 switch region as a basis for differences in IgG3 production.

High and low serum concentrations of IgG3 are associated with the human G3 m(b) and G3 m(g) allotypes, respectively. We previously hypothesized that a low frequency of switching is the most likely defect in (g) allotype-positive individuals, and therefore analyzed the structure, recombination breakpoints, and binding of nuclear proteins to the switch (S)gamma3 regions of these two allotypes. There are no allotype-associated differences in the length and basic structure of the Sgamma3, since both contain eighteen 79-bp repeats. However, we found a number of allotype-associated nucleotide changes. As in the mouse system, there is a preferential switching to the B site, or switch nuclear protein/nuclear factor-kappaB motif, with a clustering of switch breakpoints at the most 5' residue of the B site. The B site sequence used most frequently in switching was found to be mutated at this nucleotide in the (g) allotype-associated Sgamma3. This change was shown by electrophoretic mobility shift assay to alter the binding of the switch nuclear protein/nuclear factor-kappaB protein to the B site. Taken together, these data suggest that polymorphism within Sgamma3 may contribute to allotype-associated differences in IgG3 switching, and that specific sequences within the Sgamma3 79-bp repeats could be mechanistically important for switch recombination.

CD40 signaling induces interleukin-4-independent IgE switching in vivo.

T cell-deficient T cell receptor (TCR) beta-/- x TCR delta-/- knockout mice lack circulating IgE and fail to produce antigen-specific IgE in response to stimulation with T cell-dependent antigens. We show here that these animals are able to produce significant levels of circulating polyclonal IgE when injected with an agonistic anti-mouse CD40 monoclonal antibody. CD40-mediated induction of circulating polyclonal IgE in T cell-deficient mice was only partially reduced when the animals were co-treated with neutralizing anti-interleukin-4 (IL-4) antibody. The IL-4 independence of this response was further supported by experiments showing that anti-CD40 antibodies induced circulating IgE when injected into IL-4 knockout mice, and sterile RNA epsilon transcript production when cultured with purified B cells from the same mice. These data strongly suggest that CD40 signaling causes IL-4-independent IgE switching in mice.

Cell cycle regulation of immunoglobulin class switch recombination and germ-line transcription: potential role of Ets family members.

Previous studies have indicated that transcription of germ-line (GL) CH genes is necessary to obtain immunoglobulin (Ig) class switching. We report here a correlation between proliferation, switching and GL transcripts. Smu-S gamma 1 switch recombination in lipopolysaccharide (LPS) + interleukin-4 (IL-4)-activated mouse B cells was assayed by a digestion-circularization polymerase chain reaction. Switching to gamma 1 is reduced upon inhibition of DNA synthesis with hydroxy-urea (HU) or aphidicholin (AC). Incubation of activated B cells with HU severely reduces steady-state levels of GL gamma 1 and epsilon RNA. By utilizing elutriation to synchronize B cell blasts in different phases of the cell cycle, it was found that GL gamma 1 transcripts are mainly expressed in G1 and S phases, but not in G0. Using the electrophoretic mobility shift assay, we characterized two major LPS-induced complexes, which bind to the GL gamma 1 promoter and are expressed at levels which correlate with the amount of LPS-induced DNA synthesis. Furthermore, the intensity of the complexes is reduced when cells are arrested with the DNA synthesis inhibitors HU or AC. Elutriation experiments revealed that the complexes are expressed in G1 and S, but not in G0. They bind to an Ets consensus element near the major initiation sites used in proliferating cells. The possible implications of these findings for Ig isotype switching are discussed.

Homotypic aggregation of murine B lymphocytes is independent of CD23.

CD23 is a low-affinity receptor for IgE (Fc epsilon RII). Functions attributed to CD23 not involving IgE suggest that it interacts with ligands other than IgE. CD21 has recently been described as a counter ligand for CD23. A number of lines of evidence have implicated CD23 as an adhesion molecule in human B cells. We have investigated the role of CD23 in homotypic B cell aggregation in the mouse, using lipopolysaccharide plus interleukin-4-induced aggregation as a model system. In this system high levels of aggregation are accompanied by a massive up-regulation of CD23 expression. However, in contrast to what has been observed in human B cells, we find no evidence of a role for CD23 in homotypic adhesion of murine B cells.

Activation of the Ig germ-line gamma 1 promoter. Involvement of C/enhancer-binding protein transcription factors and their possible interaction with an NF-IL-4 site.

Ig isotype switching in B lymphocytes is preceded by transcription of the corresponding unrearranged, or germ-line (GL), CH gene. The promoter of mouse GL C gamma 1 transcripts has been shown to be located within a 349-bp KpnI/Bg/II fragment, extending from -147 to +202 bp relative to the first transcription initiation site. By the electrophoretic mobility shift assay, we have analyzed nuclear extracts from three B cell lines and splenic B cells for the presence of proteins binding to this fragment. We show that they give different patterns of DNA binding, implying significant complexity in the regulation of this locus. We focused on the sIgM+ mouse B lymphoma line L10A6.2 that has been shown able to confer responsiveness of the GL gamma 1 promoter to phorbol ester plus IL-4. Activation of this cell line results in altered expression of several nuclear DNA-binding complexes involving two members of the C/enhancer-binding protein (EBP) family of transcription factors, namely C/EBP beta (nuclear factor (NF)-IL-6/LAP) and Ig/EBP-1 (C/EBP gamma). The complexes bind to two C/EBP elements, one at about -115 bp and one near the first RNA start site. In normal B cells stimulated by LPS or IL-4, new complexes appear that bind to C/EBP and NF-IL-4 elements, respectively, located within the -125/-101 region. The -125/-101 segment previously has been shown to be required for transcriptional activation. We discuss these findings in relation to the presence of consensus C/EBP binding sites in other IL-4-regulated promoters.

CD23 and CD21 function as adhesion molecules in homotypic aggregation of human B lymphocytes.

We have previously found that interleukin-4 and CD40 monoclonal antibodies (mAb) are strong potentiators of homotypic B cell aggregation which is dependent on LFA-1. We show here that CD23 mAb were also able to inhibit aggregation to a similar extent as LFA-1 antibodies. This inhibition was restricted to the MHM6 epitope of CD23 and antibodies to other epitopes [Epstein-Barr virus (EBV) CS-1, EBV CS-2, EBV CS-5 and mAb 25] or occupation of the Fc-binding site by IgE had no or a slightly enhancing effect on aggregation. When testing two antibodies to CD21, the recently defined ligand for CD23, one of these (BU32) was found to be inhibitory whereas the other (THB5) had no effect. By combining antibodies to LFA-1 and CD23, aggregation was often completely inhibited. These data suggest that LFA-1/ICAM-1 and CD23/CD21 are the major molecules involved in homotypic aggregation of human B cells.

CD2: a functional adhesion molecule on murine B cells, involved in interleukin-4-induced aggregation.

The murine equivalent to CD2, previously known as a T cell marker, is expressed on mouse B cells. The monoclonal anti-CD2 antibody 12-15 was found to induce B cell homotypic adhesion. When treated with F(ab')2 fragments of 12-15, purified, resting B cells aggregate within 2 h of incubation and the response is optimal after 20 h. Anti-CD2-induced aggregation is a dose-related active process, dependent on temperature, metabolic energy and divalent cations. Aggregation is inhibited by two different Fab monomers of anti-CD2, implying that it is the CD2 molecule itself that functions as an adhesion molecule. We also report that interleukin 4-induced B cell homotypic adhesion involves CD2-mediated cell binding, since the antibodies specific for mouse, CD2, inhibited interleukin-4-induced cell aggregation.

T and B cell collaboration: induction of motility in small, resting B cells by interleukin 4.

In this report we investigate if IL 4 can work as a chemoattractant factor by inducing locomotion in B cells. We found that murine recombinant IL 4 (rIL 4) induced motile morphology and migration through polycarbonate micropore filters of murine, splenic B cells at an optimal concentration of 3 ng/ml. Kinetic studies revealed optimal migration at 8-16 h, although a significant response could be detected already after 1 h. Flow cytometric studies confirmed that the migrated cells were indeed B cells. We also compared the activity of small, dense B cells and large, low-density B cells, based on Percoll gradient separation. We found no difference in IL 4-induced motility among the two groups. Furthermore, we looked at B cells activated in vitro by preculture in lipopolysaccharide (LPS) or IL 4. Our data indicate that both LPS and IL 4 can increase the general capacity for motility in B cells after preculture for 24 h. T and B cell collaboration requires close cell-cell contacts in order for T cell help to be administered to the B cell. One way of enhancing such cell contacts could be through directional cell migration induced by helper factors (chemotaxis). We suggest that IL 4 can play a role as a chemoattractant factor that enhances cell contacts between T helper cells and B cells.

Cholera toxin acts synergistically with IL-4 to promote IgG1 switch differentiation.

Previously, we reported that cholera toxin (CT) causes LPS-stimulated membrane (m)IgM+ B cells to undergo increased switch differentiation to IgG- and IgA-producing B cells. In this study we determined whether this effect is specific for one or several of the IgG subclasses and whether B cells exposed to CT respond differently to IL-4, a lymphokine with switching capabilities. In initial studies we found that in LPS-stimulated, mIgM+ B cell cultures, CT eightfold enhanced the formation of IgG1-producing B cells, whereas it only weakly enhanced, one- to twofold, the formation of IgG3-producing B cells. In addition, CT synergistically enhanced the induction of IgG1-producing B cells by IL-4, even at plateau concentrations of IL-4. In contrast, IgM and IgG3 responses were suppressed in the CT plus IL-4-containing cultures as compared to those containing only LPS or LPS and CT. Furthermore, CT plus IL-4 had no enhancing effect on the formation of cells producing IgA; on the contrary, the presence of IL-4 led to a reversal of the stimulatory effect of CT on the IgA response. In further studies, we found that CT affected B cell differentiation at the gene level, before final gene recombination has occurred. Thus, CT together with LPS induced faint but detectable germline gamma 1 RNA transcripts not seen with cells cultured in LPS alone. However, more strikingly, CT enhanced by several-fold expression of germline gamma 1 RNA transcripts in LPS-stimulated B cell cultures containing optimal IgG1-inducing concentrations of IL-4. In addition, despite its weakly positive effect on IgG3 production. CT inhibited expression of germline gamma 3 RNA transcripts in cultures containing LPS and caused a further decrease in such transcripts in cultures containing LPS and IL-4. Finally, we found that CT enhanced the in vivo IgG1 but not the IgG3 or IgM anti-DNP serum antibody response of mice immunized with DNP-LPS. Taken together, these studies suggest that CT more strongly promotes B cell differentiation to IgG1 than to any other IgG subclass in LPS-stimulated cultures. CT acts alone or in synergy with IL-4, early in B cell differentiation to promote IgG1 expression in LPS-stimulated B cell cultures, probably by inducing early steps in the switch to this isotype such as the production of germline gamma 1 RNA transcripts.

Frequencies of interleukin-5 mRNA-producing cells in healthy individuals and in immunoglobulin-deficient patients, measured by in situ hybridization.

Interleukin-5 (IL-5) has previously been demonstrated to enhance immunoglobulin synthesis, especially IgA. Thus, it could be hypothesized that a defect production of IL-5 may cause immunoglobulin deficiency. We have analysed the frequency of IL-5 mRNA-producing cells in healthy adults and in patients with common variable immunodeficiency or selective IgA deficiency. Unstimulated lymphocytes were rarely found to synthesize IL-5 as measured by in situ hybridization. However, pokeweed mitogen and several other activating ligands induced the synthesis of IL-5 mRNA in peripheral blood and spleen lymphocyte cultures. After pokeweed mitogen activation, the number of IL-5 mRNA-producing cells most often peaked on day 3 with a maximal frequency of around 1-2% of mononuclear cells. In a kinetic study we were unable to detect any peak frequency differences between healthy controls (mean 0.44%) and 20 patients (mean 0.58%). Thus, although IL-5 has been reported to be an important regulator of IgA synthesis, a defect production does not seem to be the underlying mechanism in human immunoglobulin deficiency.

Induction of germ-line immunoglobulin heavy chain transcripts by mitogens and interleukins prior to switch recombination.

It has recently been postulated that immunoglobulin class switching is preceded by transcription from unrearranged heavy chain genes. In this report, we have investigated the conditions under which RNA transcribed from unrearranged C gamma 3, C gamma 1, C gamma 2b, C gamma 2a, C epsilon and C alpha genes are induced in normal spleen cells by mitogens and/or interleukin (IL) 4, IL 5 and interferon-gamma. Lipopolysaccharide (LPS) plus IL 4 induced germ-line gamma 1 and epsilon transcripts. LPS induced gamma 2b and gamma 3 transcripts and high doses of IL 4 suppressed these LPS-induced transcripts. Interferon-gamma induced low levels of germ-line gamma 2a transcripts and profoundly suppressed the gamma 1 and epsilon transcripts induced by LPS and IL 4. IL 5 alone or in combination with IL 4 and/or LPS did not induce germ-line alpha transcripts. Spleen cells of the partially immunodeficient mice CBA/N and C3H/HeJ, which do not express IgG3 could be induced, however, by polyclonal activators to express germ-line gamma 3 and gamma 2b transcripts. The data indicate that the capacity of a ligand to induce/suppress transcription of a particular unrearranged heavy chain gene is a good indicator of its capacity to induce switching to the corresponding Ig isotype. However, it is also clear that control of switching can be carried out at other levels.