Surface Expression Of CD23 Research Articles

Isolation of Trypanosoma brucei brucei Infection-Derived Splenic Marginal Zone B Cells Based on CD1dHigh/B220High Surface Expression in a Two-Step MACS-FACS Approach.

Magnetic- and fluorescent-activated cell sorting (MACS and FACS) are used for isolation of distinct cell populations for subsequent studies including transcriptomics. The latter allows for the analysis of infection-induced alterations in gene expression profiles. MACS and FACS both use antibodies against cell surface molecules to isolate populations of interest. Standardized methods for both approaches exist for use in mouse models. These protocols, however, do not account for the fact that infection-associated immunopathology can significantly modulate the cell surface expression of targeted molecules. This is the case for Trypanosoma brucei brucei infection, where downregulation of CD23 surface expression on B cells has been reported. This hallmark of progressing infection interferes with the commercially available MACS technique for B cell purification, as CD23 expression is the target for the separation between Marginal Zone (MZ) and Follicular (Fo) B cells. Here, we provide a robust alternative method for isolation of infection-derived MZ B cells using CD1d and B220 surface molecules in a two-step MACS-FACS approach. The method yields 99% pure viable infection-derived MZ B cells, allowing extraction of a high quality total RNA suitable for subsequent RNA sequencing.

Abstract 260: BAFF 60mer is Critical for B Cell Activation and BAFF Depletion Suppresses AAA Formation

Marginal zone and follicular B cells together constitute the B2 cell population, which is known to promote cardiovascular diseases by secretion of pathogenic antibodies. However, it is not completely understood how B2 cells are activated. Here, we tested the hypothesis that B cell activating factor (BAFF) activates B2 cells and promote abdominal aortic aneurysm (AAA) formation. Since BAFF can either exist as a 3mer or multimerize to a highly active 60mer, we further examined if the 60mer is critical for B2 cell-mediated pathogenicity. Anti-BAFF antibody (Ab) Sandy-2 was injected to C57BL/6 male mice at 1 mg/kg once in every 14 days. AAA was induced by topical elastase model after 14 days of Sandy-2 injection. Native PAGE and ELISA methods were used to determine binding of Abs to recombinant BAFF 3mer and 60mer. For in vitro experiments, B cells were isolated from murine spleens. Activation of B cells was examined by Western blotting and RNA sequencing and by surface expression of CD23 and MHC II by flow cytometry. Metabolic reprogramming of B cells by BAFF was determined by extracellular flux analysis using a Seahorse XF24 Flux Analyzer. Sandy-2 bound to both 3mer and 60mer, resulting in suppressed AAA formation (n=8, p<0.05) with (1) marked depletion of B2 cells, transitional 2, germinal center, plasma and memory B cells, but not transitional 1 and B1 cells, (2) a lower level of IgG1 and IgG2, and (3) a lack of immunoglobulin deposition in AAA sections. In vitro , the 60mer, but not the 3mer, significantly activated both NF-kB1 and -kB2 signaling, and induced expression of B2 cell activation markers and anti-apoptotic genes in B cells. Inhibitors of NF-kB signaling decreased B cell activation in response to 60mer. The 60mer treatment significantly increased mitochondrial respiration and glycolysis in B cells, supporting an activated status. An antibody against multimerization site of BAFF (anti-multiBAFF) significantly suppressed B cell activation relative to a control Ab, in a neutrophil:B cell co-culture model. The effect of the anti-multiBAFF Ab on AAA formation is currently being tested in our laboratory. Altogether, our results suggest a critical role for BAFF 60mer in skewing B cells to an activated B2 cell phenotype, supporting a pathogenic role of B2 cells in AAA.

STAT6 Is Recurrently and Significantly Mutated in Follicular Lymphoma and Enhances the IL-4 Induced Expression of Membrane-Bound and Soluble CD23

Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and remains an incurable disease for the majority of patients who present with advanced stage disease. Virtually all patients subsequently acquire treatment resistance and a third of patients ultimately develop histologically transformed disease with aggressive clinical course and poor prognosis. Thus, there is an unmet clinical need to decipher the underlying biology of this disease, and identify dysregulated pathways that may serve as therapeutic targets.In a cohort of 258 FL specimens, we identified 33 cases with STAT6 mutations (12.8%), including 15 mutations at D419. Overall, 34 out of 35 (97.1%) of STAT6 mutations were within the DNA-binding domain. MutSigCV analysis (Lawrence, Nature 2013) indicated that STAT6 was significantly mutated in FL, i.e., more commonly mutated than expected based on background mutation rate.Previously, STAT6 mutations, including D419 hotspot mutations, have been reported in other lymphomas, including primary mediastinal B-cell lymphoma (Ritz et al., Blood 2009) and FL (Yildiz et al., Blood 2015). STAT6 proteins are transcription factors that are mainly activated by interleukin-4 (IL-4). A recent study identified a subset of IL-4 producing follicular helper T cells to be involved in the survival of FL B cells (Ame-Thomas et al., Blood 2015).We compared the expression levels of selected STAT6 target genes in FL patient samples with or without STAT6 mutations. Gene expression data (nCounter, Nanostring) was available for 138 patient samples with known STAT6 mutation status. Thereof, 13 cases had STAT6 mutations, including six at D419. Among the STAT6 target genes were CD23 (and its soluble form, sCD23) and SOCS1. Both showed significantly higher expression (P<0.0001 and P=0.0016, respectively) in STAT6 mutated lymphomas. CD23 had previously been reported to be involved in B-cell growth and survival and was thus selected for further studies.CD23 immunohistochemistry was performed on 13 patient samples with known STAT6 mutation status. Five patient samples with wild-type STAT6 stained negative for CD23. In contrast, four out of eight cases with STAT6 mutations stained positive for CD23.To test if STAT6 mutations are directly linked to enhanced CD23 expression, we stably expressed wild-type (wt) STAT6, mutant STAT6 (D419G, N421K, and D519V), or empty vector in two t(14;18) positive B-lymphoma cell lines (OCI-Ly1, OCI-Ly8). Incubation of OCI-Ly1 cell lines with IL-4 (10 ng/mL for 24h) resulted in enhanced CD23 surface expression by flow cytometry (3.2-fold for D419G, 3.1-fold for N421K, and 2.3-fold for D519V), compared to STAT6 wt (N=3), whereas no significant difference was seen in the absence of IL-4. Similar results were observed in OCI-Ly8 cells.To investigate if these findings result from enhanced transcription, we performed quantitative PCR (SybrGreen). Briefly, Ct values were normalized to GAPDH reference gene and Ct values from untreated controls were subtracted from IL-4 treated samples (ΔΔCt method). These experiments showed increased CD23 transcriptional levels for OCI-Ly1 expressing mutant STAT6 (5.6-fold for D419G, 5.0-fold for N421K, and 8.4-fold for D519V), compared to STAT6 wt (N=3). For OCI-Ly8 cells, CD23 transcription was increased 4.6-fold for D419G, 5.2-fold for N421K and 3.0-fold for D519V, compared to STAT6 wt (N=3).In addition to membrane-bound CD23, the soluble CD23 (sCD23) in the microenvironment might further contribute to FL biology. To address this, we established a sCD23 ELISA using OCI-Ly1 cells stably expressing wild type or mutant STAT6. Supernatants of IL-4 stimulated cells expressing mutant STAT6 resulted in a 4.1-fold (D419G), 1.5-fold (N421K), and 1.3-fold (D519V) increase of sCD23 levels, as compared to STAT6 wt.We conclude that STAT6 is recurrently and significantly mutated in FL including hotspot mutations at D419. STAT6 mutations are gain-of-function and may promote disease progression by enhancing IL-4 induced expression of surface and soluble CD23. DisclosuresNo relevant conflicts of interest to declare.

CLL Sera Drive Maturation of Normal Monocytes to M2-like Macrophages By Direct and Indirect Mechanisms

Background. Chronic lymphocytic leukemia (CLL) is a prototypic microenvironment-dependent B-cell malignancy, in which neoplastic B cells co-evolve with a supportive tissue microenvironment to promote leukemia cell survival, growth, and drug-resistance. Within the microenvironment, hematopoietic and non-hematopoietic stromal and tumor cells produce factors that recruit circulating monocytes to tumor sites and induce differentiation into macrophages. Mirroring the Th1/Th2 paradigm, cells of monocyte-macrophage lineage reprogram their functions in response to environmental signals, undergoing M1 (classical) or M2 (alternative) activation, which represent extremes of a broad continuum of functional states. Classical M1 cells (activated by IFNs and TLR) are involved as inducer and effectors cells in polarized Th1 responses and as effectors of resistance against intracellular parasites and tumors. In contrast, M2 cells (activated by IL4 and IL13) are poor at antigen presentation, suppress Th1 adaptive immunity, actively scavenge debris, contribute to the dampening of inflammation, promote angiogenesis and tissue remodeling, and support tumor progression. One way to distinguish the two types of macrophages is based on surface antigen expression: M1-like cells up-regulate Fcg receptors I, II, III such as CD16, CD32 and CD64, whereas M2-like macrophages display abundant levels of CD23, CD163, and scavenger receptors (e.g. MCR1). Understanding the microenvironment and the crosstalk between B-CLL cells and their tissue neighbors can give insight into disease biology and for therapy.Aim. To investigate if the CLL milieu, contained within serum, influences monocyte-to-macrophage differentiation, promoting an anti (M2)- or pro (M1)- inflammatory microenvironment.Methods. Monocytes from healthy donors were isolated using Monocytes Isolation Kit II (Miltenyi) and cultured in Ultra-Low Attachment plates with 10% normal human AB serum or 10% CLL-derived serum -/+ IL4 or IFNg for 3 days. Macrophages were stained for CD23, CD64, CD32, MRC1, CD14, CD16, and data were acquired with a BD LSRII flow cytometer and analyzed by FlowJo V7.2.4 software.Results. Normal monocytes were differentiated to macrophages in vitro in the presence of sera from 24 untreated CLL patients with different prognostic factors (genomic aberrations, % CD38 and IGHVmutational status). About 45% of the CLL sera (N=10; 6 M-CLL, 4 U-CLL) drove macrophage maturation toward an M2-like phenotype, as assessed by surface expression of CD23, CD64, CD32, CD36, MRC1, etc. These 10 sera induced higher CD23 expression after 3 days in culture compared to AB human serum, whereas the levels of M1-specific markers (CD64 and CD32) did not change relative to the control. Interestingly all of these 10 CLL sera came from patients bearing 13q14 Δ (N=5), 17p13 Δ (N=3) or a combination of these (13q14 Δ + 17p13 Δ; N=1) and 17p13 Δ + trisomy12; N=1)). On the contrary, no increase in CD23 expression was detected in presence of sera from patients with 11q22 Δ (N=1) alone or in combination with 13q14 Δ (13q14 + 11q22 Δ; N=5). Of note, treatment with a neutralizing mAb specific for IL-4 did not block the CLL serum induced up-regulation of CD23 (N=2).In a parallel set of studies, normal monocytes were incubated with each of the 24 CLL sera in combination with the M1 promoting cytokine, IFNg or the M2 promoting cytokine, IL4. In all cases IL4 induced CD23 up-regulation and an M2 phenotype. Paradoxically, IFNg, which normally induces an M1 phenotype, also induced an M2 phenotype (i.e., enhanced CD23 expression) when co-cultured with sera from a subset patients (N=8; 6 M-CLL and 2 U-CLL). Of note, the IFNg stimulatory effect on CD23 expression was observed with a different set of sera from those that directly stimulated CD23 expression. Furthermore, CD64 expression did change after incubation with IFNg + CLL serum in 6 of 8 cases, yielding another unusual (CD23+CD64+) macrophage phenotype. The 2 sera that did not yield such hybrids were from M-CLL patients.Conclusions. Sera from CLL patients contain two apparently novel activities that mature normal monocytes to M2-like macrophages. The first acts directly by an action that is apparently independent of IL-4 and associates with 13q14 Δ or 17p13 Δ abnormalities. The second acts indirectly through IFNg and leads to macrophages with a hybrid M2/M1 phenotype (CD23+CD64+), suggestive of a new type of macrophage. DisclosuresNo relevant conflicts of interest to declare.

Uncoupling of Natural IgE Production and CD23 Surface Expression Levels

CD23, the low affinity receptor for immunoglobulin E (IgE), has been proposed to play a critical role in the regulation of IgE production, based on altered IgE levels in CD23-deficient mice and transgenic mouse models, as well as in mouse strains with mutations in the CD23 gene, e.g. 129 substrains. Here, we have investigated a mouse line termed LxT1 that expresses reduced CD23 surface levels on B cells, and its influence on natural IgE production. Extensive phenotypic analysis showed that CD23 surface expression was reduced in LxT1 compared to the control, without affecting B cell development in general. This CD23low surface level in LxT1 mice is not as a result of reduced CD23 mRNA expression levels or intracellular accumulation, but linked to a recessive locus, a 129-derived region spanning 28 Mb on chromosome 8, which includes the CD23 gene. Sequence analysis confirmed five mutations within the CD23 coding region in LxT1 mice, the same as those present in New Zealand Black (NZB) and 129 mice. However, this CD23low phenotype was not observed in all 129 substrains despite carrying these same CD23 mutations in the coding region. Moreover, serum IgE levels in LxT1 mice are as low as those in the C57BL/6 (B6) strain, and much lower than those in 129 substrains. These data indicate that the CD23 surface level and serum IgE level are uncoupled and that neither is directly regulated by the mutations within the CD23 coding region. This study suggests that caution should be taken when interpreting the immunological data derived from mice with different genetic background, especially if the gene of interest is thought to influence CD23 surface expression or serum IgE level.

Differential Expression and Functions of NOTCH Family Members and Involvement of NR4A1 in the Regulation of Apoptosis in CLL

AbstractAbstract 3919Chronic lymphocytic leukemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC) dependent manner linking NOTCH2 to the activated state of the leukemic cells. The transcriptional activity of NOTCH2 is associated with the expression of CD23 and enhanced CLL cell viability. However, the regulation and possible functions of the individual NOTCH family members (NOTCH1–4) in CLL cells remain to be clarified.We took advantage of targeting nuclear NOTCH2 using the recently identified NOTCH2 transactivation inhibitor gliotoxin (WO 2006/135949). We also analysed the regulation and possible function of NOTCH1–4 in PKC stimulated CLL cells using a PMA model (Hubmann et al., BJH 2010) and a microenvironment model where CLL lymphocytes were co-cultured with primary bone marrow stromal cells (BMSC) (Shehata et al., BLOOD 2010).Electrophoretic mobility shift assays (EMSA) demonstrated that gliotoxin inhibited DNA-bound NOTCH2 complexes in PMA stimulated CLL cells in parallel to increasing the rate of apoptosis (mean±SD: 67±31% in gliotoxin treated cells versus 13±14% in the untreated controls, n=21). This was associated with downregulation of CD23A mRNA expression and CD23 surface expression (mean±SD: 42±32% versus 83±17%, n=21) as assessed by RT-PCR and FACS analysis. Exceptionally, one CLL case with a recently described NOTCH1 gain of function mutation appeared to be less sensitive to gliotoxin and had a persistent high expression of CD23.We next tested whether NOTCH2 inhibition by gliotoxin is a selective process or indirectly mediated by effects on proteasome regulated apoptosis. Proteasome assays showed that gliotoxin had a minimal or no effect on the chymotrypsin like activity of the proteasome in CLL cells. In addition, the activity of the proteasome regulated transcription factor NFκB and the expression of its target genes like BCL2 and MCL1 were also not influenced by gliotoxin. These data point to the selectivity of targeting NOTCH2 signaling by gliotoxin rather than indirectly through the regulation of proteasome activity.Short term (4 hours) exposure of CLL cells revealed that NOTCH1 was equally transcribed in unstimulated and in PMA activated CLL cells. NOTCH2 was upregulated in PMA activated CLL lymphocytes whereas NOTCH4 was only weakly detectable in unstimulated CLL cells. Gliotoxin treatment resulted in the downregulation of NOTCH1, NOTCH2 and NOTCH4 mRNA expression.Interestingly, the inhibition of NOTCH2 activity by gliotoxin was associated with the concomitant induction of NOTCH3 signaling especially in the presence of PMA. This was indicated by the induced mRNA expression of NOTCH3 and its preferred target gene HEY1. Moreover, the induced transcription of HEY1 correlated with the upregulation of NR4A1, a key regulator of apoptosis in activated lymphocytes. These data may thus point to a pro-apoptotic role for NOTCH3/HEY1/NR4A1 signaling in CLL cells. The data also suggest that gliotoxin induced apoptosis is associated with differential regulation of the anti-apoptotic and pro-apoptotic arms of NOTCH signalling in CLL cells.RT-PCR revealed that NOTCH1 and NOTCH2 are the main NOTCH family members which are expressed in CLL cells under co-culture conditions with BMSC and in freshly isolated CLL cells. Exposure to gliotoxin in co-culture selectively induced apoptosis in CLL cells and led to downregulation of NOTCH1 and NOTCH2 together with upregulation of NOTCH3 mRNA expression.In summary, the data suggest that nuclear NOTCH2 activity might protect activated CLL cells from apoptosis by modulating the expression of NR4A1. The induced expression of NOTCH3 and its target gene HEY1 by gliotoxin reveals the complex role of different NOTCH family members in the regulation of apoptosis in CLL cells. Therefore, the individual NOTCH receptors may have opposite effects on CLL cell viability which should be considered in therapeutic approaches aimed to target NOTCH signaling in CLL. Disclosures:No relevant conflicts of interest to declare.

Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL

Monoclonal B cell lymphocytosis (MBL) is a hematologic condition wherein small B cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and “CLL-like” MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. Ninety-one unique MBL clones were detected: 73 CLL-like MBL (CD5+CD20dimsIgdim), 11 atypical MBL (CD5+CD20+sIg+), and 7 CD5neg MBL (CD5negCD20+sIgneg). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b, and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70, and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on 7 CLL-like MBL, and showed activation of B cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

Defective expression of CD23 and autocrine growth-stimulation in Epstein-Barr virus (EBV)-transformed B cells from patients with Wiskott-Aldrich syndrome (WAS).

WAS is an X-linked, recessive, immune deficiency syndrome, characteristically associated with lymphocyte and platelet dysfunction. Peripheral B lymphocytes from WAS patients are nonresponsive to polysaccharide antigens and show reduced numbers of cells expressing the integral membrane glycoprotein, CD23. The release of CD23 proteolytic fragments, so-called soluble CD23 (sCD23), by B lymphoblasts and EBV-transformed B cell lines has previously been described, and these fragments have been shown to stimulate autocrine growth of these cells. We have found that the surface expression of CD23 is reduced on WAS compared with control EBV-B cells. Surface CD23 levels were reduced two-fold in four WAS cell lines (group I) and nine-fold in four other lines (group II). Group II WAS cell lines also showed reduced growth rates in serum-free medium when compared with group I cell lines and EBV-B cell lines from eight normal subjects. In contrast to the group II WAS lines, group I and EBV-B cells from normal individuals produced an autocrine-growth factor activity which could be absorbed by anti-CD23 antibodies. Immunoprecipitation of sCD23 from culture supernatants confirmed that group I WAS cell lines produced less sCD23, particularly the 37K fragment which was prevalent in control EBV-B cells. Northern analysis showed that CD23 mRNA levels were increased three-fold in group I and unchanged in group II WAS compared with normal EBV-B cell lines, suggesting that decreased surface expression in WAS EBV-B cells reflects post-transcriptional events. Together these results suggest that reduced cell surface expression and aberrant proteolysis of CD23 occurs in WAS patients' B lymphocytes and may contribute to impaired immune function in these patients.

C-Rel plays a key role in deficient activation of B cells from a non–X-linked hyper-IgM patient

AbstractOur previous results demonstrated that B cells from a patient (pt1) with non–X-linked hyper-IgM syndrome (HIGM) possess an atypical CD23lo phenotype that is unaffected by CD40-mediated activation. To investigate the molecular mechanism underlying defective CD23 expression in pt1 B cells, we used lymphoblastoid cell lines that express LMP1 under the control of a tetracycline-inducible promoter (LCLtet). Our analysis revealed that the CD23lo phenotype in the pt1-LCLtet cells is a direct consequence of diminished CD23 transcription. We demonstrate a marked decrease in c-Rel–containing complexes that bind to the proximal CD23a/b promoters in pt1-LCLtet extracts, resulting from an overall lower expression of c-Rel in pt1-LCLtet cells. Analysis of c-Rel mRNA revealed relatively equal amounts in pt1-LCLtet and control LCLtet cells, indicating that diminished c-Rel protein expression is unrelated to decreased transcription. Finally, a critical role for c-Rel in CD23 regulation was demonstrated by effectively altering c-Rel expression that resulted in the direct modulation of CD23 surface expression. Collectively, these findings demonstrate that low levels of c-Rel are the underlying cause of aberrant CD23 expression in pt1 B cells and are likely to play a critical role in the pathophysiology of this form of HIGM.

Differential T Cell-Mediated Regulation of CD23 (FcεRII) in B Cells and Follicular Dendritic Cells

Differences in murine follicular dendritic cells (FDC)-CD23 expression under Th1 vs Th2 conditions prompted the hypothesis that T cells help regulate the phenotype of FDCs. FDCs express CD40, suggesting that T cell-CD40L and lymphokines may be involved in regulating FDC-CD23. To test this, highly enriched FDCs were incubated with CD40L trimer or anti-CD40 to mimic T cell signaling in the presence of IFN-gamma or IL-4. Surface expression of CD23 was determined by flow cytometry, whereas mRNA levels of CD23 and its isoforms CD23a and CD23b were independently measured by quantitative PCR. When FDCs were incubated with either CD40L trimer or agonistic anti-CD40 Ab, the expression of FDC-CD23 was increased both at the mRNA and protein levels. Moreover, engagement of FDC-CD40 enhanced mRNA levels for both CD23a and CD23b isoforms. In addition, IFN-gamma substantially enhanced CD23a and CD23b mRNA levels in CD40-stimulated FDCs. Curiously, IL-4 could also up-regulate FDC-CD23a but not -CD23b. Anti-IFN-gamma dramatically inhibited FDC-CD23 in mice immunized with CFA, whereas anti-IL-4 had only a modest inhibitory effect. In contrast with FDCs, IFN-gamma inhibited surface expression of murine B cell-CD23 as well as mRNA for B cell CD23a and -CD23b, whereas IL-4 dramatically enhanced message for both isoforms as well as protein expression. In short, CD23 was regulated very differently in FDCs and B cells. Previous studies suggest that high levels of FDC-CD23 inhibit IgE production, and this IFN-gamma and CD40L-mediated up-regulation of FDC-CD23 may explain, at least in part, why Th1 responses are associated with low IgE responses in vivo.

Sophorolipids Decrease IgE Production in U266 Cells.

RATIONALE: Sophorolipids are promising modulators of the immune response. We have previously demonstrated that sophorolipids decreased sepsis related mortality at 36 hr in vivo in a rat model of septic peritonitis by modulation of nitric oxide, adhesion molecules and cytokine production. In this study we assessed the effect of sophorolipids on IgE production to explore the anti-inflammatory effects of sophorolipid in a cellular model of allergic disease. METHODS: U266 (IgE producing myeloma cell line) were cultured in complete RPMI medium (cRPMI) +/- increasing concentrations of sophorolipids (0.1-10ug/ml) for 3 days after which levels of IgE and soluble CD23 (sCD23) protein were determined in culture supernatants (ELISA); cells were assessed for cell surface expression of CD23 and CD38 and changes in cell morphology compared with controls (cRPMI, 20% sucrose vehicle). Cell viability was determined by Trypan blue exclusion (>95%). Data are reported as mean IgE (IU/ml)+/-SE and significance between groups was determined by student's t-test. RESULTS: U266 cells cultured in cRPMI or sucrose vehicle produced high levels of IgE (520IU+/-32). Addition of sophorolipids maximally decreased IgE production at 1.0ug/ml (416+/-8SE) (p<0.01). Similar numbers of sCD23 and cell surface expression of CD23 were detected among all groups. Addition of increasing sophorolipid concentration (10ug/ml) correlated with a bimodal cell surface expression of CD38 and an increase in the percentages of plasma-like cells compared with controls (14%, 4% respectively)(p<0.05). CONCLUSIONS: Sophorolipids decrease IgE production in U266 cells, possibly through affecting plasma cell activity. This suggests that sophorolipids possess anti-inflammatory activity and may provide a novel therapy in diseases of altered IgE regulation.

Jun N-terminal kinase is essential for CD40-mediated IgE class switching in B cells

CD40 ligation activates nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases p38 and C-Jun N-terminal kinase (JNK) and causes immunoglobulin class-switch recombination (CSR) in B cells. Both NF-kappaB and p38 are important for CD40-mediated CSR. The role of JNK activation in CD40-mediated isotype switching is unknown. We sought to determine the role of JNK activation in CD40-mediated isotype switching. Splenic B cells from BALB/c mice were stimulated with anti-CD40 mAb and IL-4 or with soluble CD40 ligand in the presence or absence of SP600125, an anthrapyrazolone inhibitor of JNK. The following events were examined: IgE production by means of ELISA; S(mu)-S(epsilon) deletional switch recombination by means of digestion circularization PCR; Cepsilon germline, mature epsilon, and activation-induced deaminase (AID) transcription by means of RT-PCR; and proliferation by tritiated thymidine incorporation and surface expression of CD23, CD54, and CD86 by means of FACS analysis. SP600125 at 10 microM drastically inhibited JNK phosphorylation but had little effect on CD40-mediated p38 phosphorylation and expression of the NF-kappaB dependent genes c-Myc and bcl-xL. SP600125 inhibited IgE synthesis by approximately 88% but had no effect on B-cell proliferation and survival in response to anti-CD40 + IL-4 or on upregulation of CD23, CD54, and CD86 in response to CD40 ligation. Analysis of molecular events involved in IgE class switching revealed that SP600125 had no effect on the expression of C(epsilon) germline and AID transcripts. In contrast, SP600125 severely reduced S(mu)-S(epsilon) switch recombination and expression of mature epsilon transcripts. These results demonstrate that JNK activation is essential for CD40-mediated CSR to IgE and suggest that JNK is important for AID activity in B cells.

TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology.

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r(-/-) mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r(-/-) mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r(-/-) B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r(-/-) mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.

Expression of IL13 receptor alpha 1 in human B cells committed to produce IgE

Rationale Our previous studies have shown that human B lymphocytes, stimulated to synthesize IgE, produce IL-13 as an autocrine growth factor. Little is known about the expression of IL-13 receptor alpha 1 (IL-13Rα1) during the development of IgE secreting B cells. Methods Purified B cells were isolated from human tonsils and cultured with or without anti-CD40 antibody (anti-CD40, 1 μg/ml), recombinant human IL4 (400 U/ml), ± IL13 neutralizing antibody (3 μg/ml). Cells were serially harvested during 14 day culture. IL-13Rα mRNA expression was evaluated by real time-PCR, and IL-13Rα1, IL-4Rα; and CD23 surface expression was examined by flow cytometry and laser scanning microscopy. Results IL13Rα1 mRNA was expressed at very low levels prior to stimulation, but increased following addition of anti-CD40. Over a 14 day culture period, surface IL13Rα1 was detected at low levels (MFI 65.4 ± 29.8) in unstimulated B cells, but increased significantly at 1, 5, 10, and 14 days after anti-CD40/IL4 stimulation (MFI 249.6 ± 56.5 at day 10), P<0.01 for all time points). In contrast, IL4Rα was highly expressed in unstimulated B cells, and did not significantly increase after stimulation. CD23 expression increased following CD40/IL4 stimulation ( P<0.01) and was co-expressed on cells that expressed IL-13Rα1. Anti-IL13 treatment had no effect on either IL13Rα1 or CD23 expression following stimulation. Conclusions IL13Rα1 and IL4Rα are not equally expressed in resting B lymphocytes. IL13Rα1 and CD23 are highly inducible following B cell activation. These data for the first time demonstrate the kinetics of IL-13Rα1 expression on human B cells.

CD23 defines two distinct subsets of immature B cells which differ in their responses to T cell help signals.

Transitional immature B cells undergo apoptosis and fail to proliferate in response to BCR cross-linking, thus representing a target for negative selection of potentially autoreactive B cells in vivo. In agreement with recent reports, transitional B cells were divided into developmentally contiguous subsets based on their surface expression of CD23. When transferred, CD23(+) transitional B cells readily localized to the splenic follicles and the outer PALS. Compared with CD23(-) transitional B cells, CD23(+) transitional B cells proliferated more vigorously and were rescued from BCR-induced apoptosis to a greater degree, by T cell help signals. However, both CD23(-) and CD23(+) transitional B cells failed to up-regulate CD86 (B7-2) in response to BCR ligation. These findings demonstrate that phenotypically defined subsets within the transitional B cell population are functionally distinct. Specifically, responsiveness to T cell help is a late acquisition corresponding to the stage when the B cells gain access to peripheral compartments enriched in antigen and activated T cells. The failure of transitional B cells to up-regulate CD86 to BCR-mediated stimulation suggests a unique interaction between transitional B cells and T cells with implications for tolerance in the T cell compartment.

Signal transduction pathways triggered by the FcϵRIIb receptor (CD23) in human monocytes lead to nuclear factor-κB activation

Background: Alveolar macrophages play a key role in the initiation of the inflammatory reaction of allergic asthma. Alveolar macrophages and peripheral blood monocytes are activated when IgE/allergen immune complexes bind to the CD23 receptor, which leads to the production of inflammatory cytokines. Objective: We sought to investigate the molecular mechanisms regulating this early inflammatory response. We have focused on the study of the signal transduction pathways triggered by CD23 in human monocytes and the promonocytic cell line U937. Methods: CD23 was cross-linked in human monocytes and U937 cells with IgE immune complexes. Surface expression of CD23 was determined by FACS analysis. Transcription factor activation and gene transcription were studied by gel-shift assays and Northern blot analysis, respectively. IκBα phosphorylation and degradation was analyzed by Western blot. Results: Nuclear factor (NF)-κB is the main transcription factor involved in the gene activation that follows CD23 cross-linking in monocytes. CD23-induced NF-κB is a heterodimer composed of p65/p50 subunits. NF-κB nuclear translocation is secondary to the phosphorylation and subsequent degradation of the NF-κB inhibitory molecule IκBα. Tyrosine kinase–dependent, and not protein kinase C–dependent, pathways mediate CD23-triggered NF-κB activation but do not participate in the direct phosphorylation of IκBα. IκBα degradation and NF-κB nuclear translocation correlate with transcriptional activation of the inflammatory cytokines TNF-α and IL-1β. Conclusions: NF-κB is the main transcription factor involved in the signal transduction pathway of CD23 in monocytes. (J Allergy Clin Immunol 1999;104:376-87.)

Upregulated surface expression of intracellularly sequestered Igepsilon receptors (FcepsilonRII/CD23) following activation in human peripheral blood eosinophils.

We investigated the regulation, secretion, and surface expression of the low-affinity FcepsilonRII receptor (CD23) in eosinophils isolated from human blood using multiple monoclonal antibodies (mAbs) directed at different epitopes of human CD23. Substantial surface expression of CD23 was not demonstrated in the resting state. Mean fluorescence intensity (MFI) measured by flow cytometry was 7. 1 +/- 0.8 for 9P25 mAb (p = NS) and 15.7 +/- 3.8 for BU38 mAb (p <. 04) versus 5.3 +/- 1.0 for IgG1 isotype control Ab. By contrast, MFI using BU38 mAb was 154 +/- 18 for JY-B lymphocytes (p <.0001 versus eosinophils). Despite weak surface expression, eosinophil permeabilization demonstrated substantial intracellular expression of CD23; MFI was 33.6 +/- 5.2 for 9P25 mAb versus 4.4 +/- 0.43 for IgG control (p <.001). Western blot analysis using both positive and negative controls demonstrated immunological identity with CD23 on JY-B lymphocytes. Activation of eosinophils caused rapid translocation of CD23 to the surface membrane (160 +/- 33 MFI; p <. 005), which was maximal within 30 sec. Secretory CD23 was detected within the perfusate also at 30 sec and was fully reinternalized at 10 min. This is the first demonstration of the presence of intracellular CD23 in human eosinophils. Our data indicate that eosinophils rarely express CD23 on their surface but are capable of transient high-level expression and secretion with rapid reuptake of intracellular stores of CD23.

Role of JAK3 in CD40-Mediated Signaling

CD40 is a member of the tumor necrosis factor receptor family and plays an important role in B-cell survival, growth, differentiation, and isotype switching. Recently, CD40 has been shown to associate with JAK3, a member of the family of Janus Kinases, which are nonreceptor protein kinases involved in intracellular signaling mediated by cytokines and growth factors. To investigate the role of JAK3 in CD40-mediated signaling, we studied the effect of CD40 stimulation on B-cell proliferation, IgE isotype switching, and upregulation of surface expression of CD23, ICAM-1, CD80, and LT-α in JAK3-deficient patients. Our studies show that stimulation of B cells with monoclonal antibody to CD40 in the presence of interleukin-4 (IL-4) or IL-13 resulted in similar responses in JAK3-deficient patients and normal controls. This suggests that JAK3 is not essential for CD40-mediated B-cell proliferation, isotype switching, and upregulation of CD23, ICAM-1, CD80, and LT-α surface expression.

Role of JAK3 in CD40-Mediated Signaling

CD40 is a member of the tumor necrosis factor receptor family and plays an important role in B-cell survival, growth, differentiation, and isotype switching. Recently, CD40 has been shown to associate with JAK3, a member of the family of Janus Kinases, which are nonreceptor protein kinases involved in intracellular signaling mediated by cytokines and growth factors. To investigate the role of JAK3 in CD40-mediated signaling, we studied the effect of CD40 stimulation on B-cell proliferation, IgE isotype switching, and upregulation of surface expression of CD23, ICAM-1, CD80, and LT-α in JAK3-deficient patients. Our studies show that stimulation of B cells with monoclonal antibody to CD40 in the presence of interleukin-4 (IL-4) or IL-13 resulted in similar responses in JAK3-deficient patients and normal controls. This suggests that JAK3 is not essential for CD40-mediated B-cell proliferation, isotype switching, and upregulation of CD23, ICAM-1, CD80, and LT-α surface expression.

Activation of B-lymphocytes during pollen season. Effect of immunotherapy.

B-lymphocytes play an important part in the allergic reaction as producers of IgE antibodies. To investigate the cell surface expression of the activation antigens CD23, CD40 and HLA-DR on B-lymphocytes in birch pollen allergic patients before and during birch pollen season and to study the effect of immunotherapy. The study included 24 birch pollen allergic patients half of whom were treated with immunotherapy against birch pollen before the start of the season. Eleven of the 24 patients had asthma. Blood samples were taken and lung function was registered before the season began and before the immunotherapy treatment in January to February and during the season in May. The relative number of B-lymphocytes (CD19+) of the lymphocyte population and the cell surface expression of CD23, CD40 and HLA-DR on B-lymphocytes was measured by the use of flow cytometry. In the control group of patients the relative number and concentration of B-lymphocytes, the cell surface expression of CD23, CD40 and HLA-DR on B cells, and the serum concentration of IgE increased during season compared with before season. In contrast, in the immunotherapy treated patients no changes in the number of B cells or cell surface expression of CD23, CD40 and HLA-DR were demonstrated. The elevated expression of CD23, CD40 and HLA-DR on B cells, combined with increased levels of IgE in allergic patients during season could be explained by the effect of cytokines produced by activated TH2 cells. A shift from TH2 to TH1 cells might be the mechanism after the absence of signs of B-cell activation in immunotherapy treated patients. The prevention of increased cell surface expression on B cells by immunotherapy may constitute a significant mechanism behind the beneficial effects of immunotherapy in the treatment of pollen atopy.