Expression and localization of rabbit B-cell activating factor (BAFF) and its specific receptor BR3 in cells and tissues of the rabbit immune system

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Abstract

Rabbits are widely used for vaccine development, and investigations of human infectious and autoimmune diseases such as Systemic Lupus Erythematosus (SLE). For these applications, we cloned, sequenced and expressed rabbit B-cell Activating Factor (BAFF), and localized BAFF in cells and tissues of the rabbit immune system. The rabbit homolog of the human BAFF binding site (miniBR3 peptide) within the BAFF-specific receptor BR3 was synthesized. This 26-residue core domain binds to recombinant rabbit BAFF protein. Flow cytometric analyses using purified recombinant rabbit BAFF combined with real-time PCR findings revealed that BAFF detected on peripheral blood B-cells from normal rabbits is probably complexed to BAFF receptors rather than produced by the B-cells. BAFF was detected in developing appendix of young rabbits by immunohistochemical staining suggesting that BAFF plays a role during the period following birth when rabbit B-cell development and pre-immune antibody repertoire diversification and selection is occurring.

Introduction

Members of the TNF/TNF-R superfamily have been shown to play significant roles in B-cell differentiation. B-cell activating factor of the TNF family (BAFF; also termed TNFSF13b, BLyS, TALL-1, and zTNF4) is a TNF-like cytokine that is essential for the survival and homeostasis of B lymphocytes [1], [2], [3] (reviewed in [4] and cited articles). BAFF is a type II transmembrane protein but also becomes a soluble ligand after cleavage at the cell surface by a furin-like protease. BAFF was initially found to be expressed and secreted by cells of the myeloid lineage [5], but there have been recent reports that BAFF is also expressed by neutrophils [6], neoplastic B-cells [7], [8], activated mouse B-cells [9], T-cells from patients with autoimmune disorders [10], as well as by non-hematopoietic cells including astrocytes [11] and fibroblast-like synoviocytes of mesenchymal origin [12]. These mesenchymal-derived cells were shown to express functional BAFF in vitro after induction with proinflammatory cytokines such as IFN-γ and TNF-α [12]. Although BAFF is essential for development and maintenance of most B-cells, CD5-positive B-1 cells develop in mice lacking either BAFF or BAFF-receptor (BR3) [13], [14]. Since BAFF was dispensable for B-1 B-cell development in mice, we investigated whether functional BAFF and it receptors are present in rabbits where the majority of B-cells are CD5 positive [15]. We report here, comparative sequence and expression analyses of rabbit BAFF, and BR3 and production of recombinant BAFF (rBAFF) protein.

BAFF can bind to three distinct receptors, the BAFF receptor (BR3, BAFF-R), the transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and the B-cell maturation Ag (BCMA)[16], [17]. BR3 binds BAFF with high affinity and is considered the only BAFF-specific receptor [18]. Failure of survival of peripheral B-cells from immature transitional to mature naïve stages is observed in mice lacking BAFF or with defective BR3 [19]. TACI and BCMA bind BAFF with intermediate and low affinity, respectively. TACI plays a key role in negatively regulating mature B-cell homeostasis, but TACI also plays an important role in T-cell independent B-cell responses and class switch recombination [20], [21]. Much less is known about BCMA, but recent reports suggest a role of BCMA in controlling the lifespan of long-lived plasma cells [22]. All three receptors are mainly expressed on B-cells, but their expression levels change with B-cell maturation.

The early development of B-cells in the rabbit initiates in sites including fetal liver, omentum and bone marrow (reviewed in Mage et al.) [23]. After birth, the pre-immune antibody (Ab) repertoire is expanded and diversified in gut-associated lymphoid tissue (GALT). The molecular events that occur during Ab diversification have been intensively studied in rabbit, but less is known about the control at functional checkpoints and B-cell survival.

The present report provides the basis for further studies of the function of BAFF and its receptors in rabbit immune responses that are particularly needed now because in addition to being a valuable resource for development of diagnostic and therapeutic antibodies, rabbits are important for vaccine development and as animal models of human diseases. Our laboratory has described a model of the autoimmune disease SLE in allotype-defined pedigreed rabbits [24], [25] and (Yang J, et al., ms in preparation). Before we could investigate the specific roles of BAFF and its receptors in rabbit B-cell development, maturation and Ab production in normal and autoimmune animals, it was necessary to first determine their cDNA and encoded protein sequences and their expression patterns in hematopoietic cells. In this study, we used a cross-species comparison strategy to design PCR primers, amplify, clone, and sequence rabbit BAFF and BR3. We found BAFF protein in developing appendix of young rabbits suggesting that BAFF plays a role during the period following birth when B-cell development, pre-immune Ab repertoire diversification, selection and expansion is occurring. We quantitated BAFF and BR3 mRNA expression and detected staining patterns on peripheral blood mononuclear (PBMC) and spleen cells of adult rabbits, expressed recombinant rabbit BAFF protein and utilized the protein for further investigations of BAFF and BR3 interactions.

Section snippets

Animals

Rabbits were obtained from the allotype-defined pedigreed colony maintained at the National Institute of Allergy and Infectious Diseases. The animal studies described here were reviewed and approved by the animal care and use committees of NIAID/NIH (ASP LI6) and of the Spring Valley Laboratories, Inc. where the NIAID allotype-defined rabbit colony was housed. Normal adult rabbits of known Ig allotypes from our breeding colony, provided whole blood and spleens for flow cytometry,

Rabbit BAFF and BR3 cloning and sequence comparison

The full-length rabbit BAFF, part of BR3 cDNA sequences obtained by reverse transcriptase PCR and the conceptual amino acid sequences deduced from the cDNA sequences are shown in Figs. 1A and B and 2. At the protein level, full-length rabbit BAFF is five amino acids longer than human BAFF and there are two potential N-linked glycosylation sites (asterisks in Fig. 1A) similar to those found in human BAFF. Because rabbit BAFF protein also contains a furin-like protease site (arrow in Fig. 1A),

Discussion

BAFF and its receptors are important for B-cell homeostasis. Discovery of the BAFF system has provided immunologists with new insight into the mechanisms that control B-cell survival during maturation in the periphery. Although BAFF is a beneficial factor that promotes B-cell survival and enhances immune responses, excessive BAFF production seems to be able to subvert B-cell tolerance. Thus BAFF and its receptor expression must be carefully regulated, and decoy receptors based on TACI, BCMA,

Acknowledgements

This research was supported by the intramural research program of the NIH, NIAID and the Oak Ridge Associated Universities. We thank Drs. Alejandro Schaffer and Richa Agarwala, NCBI for identification and assistance with sequence analyses of a rabbit BR3-containing BAC clone, Dr. Jan Lukszo, Peptide Synthesis and Analysis Laboratory, Research Technology Branch, NIAID for synthesis of rabbit miniBR3, Thomas Hofer and Christoph Rader for help with BAFF protein expression, Rami Zahr, Jacqueline

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