Patty Zwollo

B Cell Heterogeneity in the Teleost Kidney: Evidence for a Maturation Gradient from Anterior to Posterior Kidney

The fish immune system is quite different from the mammalian system because the anterior kidney forms the main site for hematopoiesis in this species. Using transcription factor-specific Abs derived from the murine system, together with anti-trout Ig Abs and Percoll gradient separation, we analyzed B cells from trout kidney sections and compared them to those from spleen and blood. For this study, immune cells were separated by Percoll gradients, and the resulting subpopulations were defined based on expression of B cell-specific transcription factors Pax-5 and B lymphocyte-induced maturation protein-1, as well as proliferative and Ig-secreting properties. Comparison of kidney, blood, and spleen B cell subsets suggest that 1) the anterior kidney contains mostly proliferating B cell precursors and plasma cells; 2) posterior kidney houses significant populations of (partially) activated B cells and plasmablasts; and 3) trout blood contains resting, non-Ig-secreting cells and lacks plasma cells. After LPS induction of resting B cells in vitro, the kidney and spleen have a high capacity for the generation of plasma cells, whereas the blood has virtually none. Our results indicate that trout B cell subsets are profoundly different among blood, anterior kidney, posterior kidney, and spleen. We hypothesize that developing B cells mature in the anterior side of the kidney and then migrate to sites of activation, either the spleen or the posterior kidney. Lastly, our data support the notion that the trout kidney is a complex, multifunctional immune organ with the potential to support both hemopoiesis as well as humoral immune activation.

Plasmablast and Plasma Cell Production and Distribution in Trout Immune Tissues

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.

Molecular and cellular analysis of B cell populations in the rainbow trout using Pax5 and immunoglobulin markers

To date, the trout B-cell is poorly defined, as many essential molecular markers are not yet available for this species. In mammalian systems, the transcription factor Pax5, expressed from pre-B through plasmablast stages, provides an important marker for B-cell differentiation. In a previous study we showed that Pax5 is expressed in the trout. Here we identify trout B-cell populations that vary in expression of Pax5, membrane and secreted Ig. Immune tissues were separated based on concentration of surface IgM, and analyzed by qPCR and flow cytometry. Results suggest that spleen and PBL contain mostly resting B cells, which lack secreted Ig. While the great majority of splenic B cells become strongly activated upon LPS stimulation, PBLs do not. Additionally, anterior kidney contains both developing B and Ig-secreting B-cell populations, but few resting, mature B cells. Lastly, posterior kidney contains multiple B-cell populations in various states of activation. We conclude that trout immune tissues contain multiple, developmentally diverse and tissue-specific B-cell populations as defined by their relative expression of Pax5, surface IgM, and secreted IgM.

B Cell-specific Activator Protein Prevents Two Activator Factors from Binding to the Immunoglobulin J Chain Promoter until the Antigen-driven Stages of B Cell Development

The immunoglobulin J chain gene is inducibly transcribed in mature B cells upon antigen recognition and a signal from interleukin-2 (IL-2). B cell-specific activator protein (BSAP), a transcription factor that silences J chaintranscription, has been identified as a nuclear target of the IL-2 signal. The levels of BSAP progressively decrease in response to IL-2 and this change correlates with the differentiation of B cells into antibody secreting plasma cells. Here we report the binding of the upstream stimulatory factor (USF) to an E-box motif immediately upstream from the BSAP site on the J chain promoter. Mutations in the USF binding motif significantly decrease J chain promoter activity in J chain expressing B cell lines. We also show that a functional relationship exists between USF and a second J chain positive-regulating factor, B-MEF2, using co-immunoprecipitation assays and transfections. Finally, we provide evidence that the binding of BSAP prevents USF and B-MEF2 from interacting with the J chain promoter during the antigen-independent stages of B cell development. It is not until the levels of BSAP decrease during the antigen-driven stages of B cell development that both USF and B-MEF2 are able to bind to their respective promoter elements and activate J chaintranscription.

CD40 stimulation induces Pax5/BSAP and EBF activation through a APE/ref-1-dependent redox mechanism

CD40 is a member of the growing tumor necrosis factor receptor family that has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154, mainly expressed on activated T cells, stimulates B cell proliferation, differentiation, isotype switching, up-regulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. In this study we demonstrate that the redox factor APE/Ref-1 acts as a key signaling intermediate in response to CD40-mediated B cell activation. The transcription factors Pax5a or BSAP (B cell lineage-specific activator protein) and EBF (early B cell factor) are constitutively expressed in spleen B cells and CD40 cross-linking induces increases in Pax5a and EBF binding activity compared with nonstimulated B cells. We show that upon CD40 antibody-mediated cross-linking, APE/Ref-1 translocates from the cytoplasm to the nucleus of activated B cells, where it modulates the DNA binding activity of both Pax5a and EBF. Moreover, we show that the repression of APE/Ref-1 protein production is able to block CD40-mediated Pax5a activation. We also provide evidence that APE/Ref-1 can modulate the cooperative activation of the blk promoter operated by Pax5a and EBF and that APE/Ref-1 might directly regulate EBF functional activity. Finally, we show that the interaction between Pax5a and EBF enhances EBF binding activity to its consensus sequence, suggesting that Pax5a can physically interact with EBF and modulate its DNA binding activity.

DISSECTING TELEOST B CELL DIFFERENTIATION USING TRANSCRIPTION FACTORS

B cell developmental pathways in teleost fishes are poorly understood. In the absence of serological reagents, an alternative approach to dissecting teleost B cell development is to use transcription factors that are differentially expressed during B cell development. This review discusses the structure and function of six transcription factors that play essential roles during teleost B cell development: Ikaros, E2A, EBF, Pax5, Blimp1, and XbpI. Research on alternative splicing of both the Ikaros and Pax5 genes in rainbow trout is presented, including their functional significance. An application is discussed that should aid in elucidating teleost B cell development and activation, by using transcription factors as developmental markers in flow cytometric analysis. Possible future studies in teleost B cell development are suggested in the context of gene regulation. Lastly, broader impacts and practical applications are discussed.

Decreased Immune Response in Zebra Finches Exposed to Sublethal Doses of Mercury

Mercury (Hg) is a ubiquitous contaminant with deleterious effects on many wildlife species. Most studies to date have focused on fish-eating birds and mammals because much historical Hg pollution is aquatic. Recently, however, comparable blood-Hg levels have been found in terrestrial insectivorous songbirds. As a result, research is needed to clarify the effects of Hg exposure on songbirds. One fundamental end point that is still poorly understood is the effect of Hg on the songbird immune system. If Hg affects the functioning of the immune system, exposed songbirds may be less able to mount an appropriate immune response against invading pathogens. To gain insight into how Hg affects songbird immune function on a cellular level, a flow cytometric assay was developed to measure lipopolysaccharide-induced B-lymphocyte proliferation in zebra finches (Taeniopygia guttata). This is the first experimental (dosing) study of the potential effect of Hg on songbird immune system functioning. Decreased B cell proliferation was observed after lipopolysaccharide exposure in individuals with greater concentrations of Hg in their blood and tissues. In addition, these individuals had decreased ratios of proliferating-to-resting B cells. This decrease in lymphocyte proliferation in response to an effective mitogen suggests that environmental exposure to sublethal levels of Hg may inhibit or delay B cell proliferation in songbirds, potentially increasing susceptibility to disease and decreasing survivorship.

The Transcription Factor NF-κB/p50 Interacts with the blkGene during B Cell Activation

The B cell-specific transcription factor Pax-5 has been shown previously to interact with the promoter of theblk gene in vitro. blk encodes a tyrosine kinase associated with the B cell receptor, which is expressed during the early but not the final stages of B cell development. To investigate whether Pax-5 regulates expression of the blkgene in vivo during B cell development and/or activation,Pax-5a was overexpressed in B cell lines. Increases inblk promoter activity using a chloramphenicol acetyltransferase reporter gene system suggested a role for Pax-5a as a transcriptional activator. Subsequent site-specific mutagenesis studies showed that mutations of the Pax-5 binding site on blksignificantly alter promoter activity, although results suggested that other factors could bind to this region as well. Using mobility shift assays, we detected an inducible transcription factor that interacts strongly with a sequence overlapping the Pax-5 site on theblk promoter and identified this as a homodimer of NF-κB/p50, a member of the NF-κB/Rel family of transcription factors. This factor was present at high levels in lipopolysaccharide-activated normal B cells and in plasma cell lines but either at low levels or undetectable levels in resting normal B cells or pre-B or mature B cell lines. In contrast, lipopolysaccharide induction of a pre-B cell line (703/Z) induced a complex that contained both NF-κB/p50 and p65. These studies suggest that different NF-κB complexes are able to interact with a sequence overlapping the Pax-5 site on the blk promoter and that the relative levels of “bound” factor influence levels of blk expression. Since p50 homodimers and p50/p65 heterodimers of the NF-κB complex should have opposing effects on blk transcription, this could provide a mechanism to differentially regulate blkexpression during B cell development and activation.

Defining terminally differentiating B cell populations in rainbow trout immune tissues using the transcription factor XbpI.

The nature of antibody-secreting cells in the rainbow trout is poorly defined. Here we describe a flow cytometric approach to help differentiate between four major trout B cell subsets present during terminal B cell differentiation: resting B cells, activated B cells, plasmablasts, and plasma cells. To aid in the identification of B cell subsets, the LPS-inducible transcription factor XbpI-S was used as a marker. An antibody specific to the stable form of inducible transcription factor X-box protein I (XbpI) was generated, which detects XbpI-S protein expression for species within the Oncorhyncus genus, including rainbow trout. Combinatorial expression patterns, or B cell signatures, were established using antibodies to XbpI-S, Pax5, and IgM in combination with a proliferation marker. We show that XbpI-S induction in trout splenic B cells increases throughout a 10-day in vitro LPS-induction period and that increased XbpI-S expression correlates with increased HCmu expression in the cell. PBLs displayed a lower level of XbpI-S induction during this incubation period, compared to spleen. We conclude that trout B cells follow a highly conserved B cell activation pathway, albeit slower than what has been observed in mammalian species. The use of XbpI-S as an activation marker for trout humoral immune activation promises to be useful for future in vivo studies, and can be applied to a broad range of teleost species.

Reduction in DNA Binding Activity of the Transcription Factor Pax-5a in B Lymphocytes of Aged Mice

Aging has been associated with intrinsic changes of the humoral immune response, which may lead to an increased occurrence of autoimmune disorders and pathogenic susceptibility. The transcription factor Pax-5 is a key regulator of B cell development. Pax-5a/B cell-specific activator protein and an alternatively spliced isoform, Pax-5d, may have opposing functions in transcriptional regulation due to the lack of a transactivation domain in Pax-5d. To study B cell-specific changes that occur during the aging process, we investigated expression patterns of Pax-5a and 5d in mature B cells of young and aged mice. RNase protection assays showed a similar transcriptional pattern for both age groups that indicates that aging has no affect on transcription initiation or alternative splicing for either isoform. In contrast, a significant reduction in the DNA binding activity of Pax-5a but not Pax-5d protein was observed in aged B cells in vitro, while Western blot analyses showed that similar levels of Pax-5a and 5d proteins were present in both age groups. The observed decrease in Pax-5a binding activity correlated with changes in expression of two Pax-5 target genes in aged B cells. Expression of the Ig J chain and the secreted form of Ig μ, which are both known to be suppressed by Pax-5a in mature B cells, were increased in B cells of aged mice. Together, our studies suggest that changes associated with the aging phenotype cause posttranslational modification(s) of Pax-5a but not Pax-5d, which may lead to an abnormal B cell phenotype in aged mice, associated with elevated levels of J chain, and secretion of IgM.