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BLyS: Member of the Tumor Necrosis Factor Family and B Lymphocyte Stimulator

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Science  09 Jul 1999:
Vol. 285, Issue 5425, pp. 260-263
DOI: 10.1126/science.285.5425.260

Abstract

The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-γ. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.

A 285–amino acid protein was identified in a human neutrophil-monocyte–derived cDNA library that shared identity within its predicted extracellular receptor-binding domain to APRIL (28.7%) (1), TNFα (16.2%) (2), and lymphotoxin-α (LT-α) (14.1%) (Fig. 1A) (3). This cytokine has been designated B lymphocyte stimulator (BLyS) on the basis of its biological activity. Analyses of the BLyS protein sequence have revealed a potential transmembrane spanning domain between amino acid residues 47 and 73 that is preceded by nonhydrophobic amino acids, suggesting that BLyS is a type II membrane-bound protein (4). Expression of this cDNA in mammalian cells [HEK 293 and Chinese hamster ovary (CHO)] and Sf9 insect cells identified a soluble form, 152 amino acids in length, with an NH2-terminal sequence beginning with Ala134(arrow in Fig. 1A). Reconstruction of the mass-to-charge ratio defined a mass for BLyS of 17,038 daltons, a value consistent with that predicted for this 152–amino acid protein with a single disulfide bond (17037.5 daltons). BLyS has been mapped to human chromosome 13q34 (5).

Figure 1

Sequence and expression pattern of human BLyS (28). (A) Amino acid sequence of BLyS and alignment with TNF family members. Shaded boxes indicate shared residues between family members. The predicted membrane-spanning region is indicated and the site of cleavage depicted with an arrow. Sequences overlaid with lines (labeled A through H) represent predicted β-pleated sheet regions. (B) Expression of BLyS mRNA. Northern hybridization analysis was performed using the BLyS open reading frame as a probe for polyadenylate-selected RNA from the indicated sources. PBMC, peripheral blood mononuclear cells. (C) BLyS expression increases following activation of human monocytes by IFN-γ. Flow cytometric analysis of BLyS expression on cultured monocytes using BLyS specific mAb (2E5) (solid lines) or an isotype matched control (IgG1) (dashed lines). Hybridomas and monoclonal antibodies were prepared as described (29–32).

The expression profile of BLyS was assessed by Northern blot and flow cytometric analyses. BLyS is encoded by a single 2.6-kb mRNA expressed in peripheral blood mononuclear cells, spleen, lymph node, and bone marrow (Fig. 1B). Lower expression was detected in placenta, heart, lung, fetal liver, thymus, and pancreas. BLyS mRNA was also detected in HL-60 and K-562, but not in Raji, HeLa, or MOLT-4 cells. Surface expression was analyzed by flow cytometry with the BLyS-specific monoclonal antibody (mAb) 2E5. BLyS was not detected on T- or B-lineage cell lines, but was restricted to cells of myeloid origin, including K-562, HL-60, THP-1, and U-937 (6). Analyses of normal blood cell types showed expression on resting monocytes that was upregulated four times after exposure of cells to interferon-γ (IFN-γ) (100 U/ml) for 3 days (Fig. 1C). A concomitant increase in BLyS-specific mRNA was also detected by quantitative polymerase chain reaction using a TaqMan machine (Perkin-Elmer Applied Biosystems) (6). BLyS was not expressed on freshly isolated blood lymphocytes or on activated T cells [anti-CD3 mAb + interleukin-2 (IL-2)], B cells (SAC + IL-2), or NK cells (IL-2 + IL-12) (6).

Purified recombinant BLyS (rBLyS) was assessed for its ability to induce activation, proliferation, differentiation, or death in numerous cell-based assays involving B cells, T cells, monocytes, natural killer (NK) cells, hematopoietic progenitors, and a variety of cell types of endothelial and epithelial origin. A biological response to BLyS was observed only among B cells in a standard costimulatory proliferation assay in which purified tonsillar B cells were cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human immunoglobulin M (IgM) as priming agents (7, 8). The rBLyS induced a concentration-dependent proliferation of tonsillar B cells similar to that of recombinant IL-2 (rIL-2) (Fig. 2A). BLyS also induced B cell proliferation when cultured with cells costimulated with graded doses of anti-IgM (Fig. 2B). A concentration-dependent response was readily observed as the amount of cross-linking agent increased in the presence of a fixed concentration of either IL-2 or rBLyS.

Figure 2

BLyS is a potent B lymphocyte stimulator. (A) The biological activity of BLyS was assessed in a standard B lymphocyte costimulation assay (33) (▴, SAC + IL-2; ▪, SAC + BLyS). (B) Proliferation of tonsillar B cells with BLyS and costimulation with anti-IgM [▪, anti-IgM only; ▴, anti-IgM + IL-2 (100 ng/ml); ▾, anti-IgM + BLyS (100 ng/ml)].

Biotinylated BLyS protein (which retained biological function in the standard B cell proliferation assays) (6) was used to assay for receptor expression. Lineage-specific analyses of human peripheral blood cells indicated that binding of biotinylated BLyS was undetectable on T cells, monocytes, NK cells, and granulocytes as assessed by CD3, CD14, CD56, and CD66b, respectively (Fig. 3A). Activation of normal human T cells with anti-CD3 mAb and IL-2 did not induce BLyS receptor expression (6). In contrast, biotinylated BLyS bound peripheral CD20+ B cells. Receptor expression was also detected on the B cell tumor lines REH, ARH-77, Raji, Namalwa, RPMI-8226, and IM-9 but not any of the myeloid-derived lines tested, including THP-1, HL-60, K-562, and U-937 (Fig. 3B). Thus, BLyS displays a B cell tropism in both its receptor distribution and biological activity.

Figure 3

BLyS receptor expression among normal human peripheral blood nucleated cells and tumor cell lines. (A) Human peripheral blood nucleated cells were obtained from normal volunteers and isolated by density gradient centrifugation. Cells were stained with biotinylated BLyS followed by PE-conjugated streptavidin and fluorescein isothiocyanate (FITC)– coupled mAbs specific for CD3, CD20, CD14, CD56, and CD66b. (B) BLyS binding to U-937 and the myeloma line IM-9. Similar results were also obtained using a biologically active FLAG-tagged BLyS protein instead of the chemically modified biotin-BLyS (6).

To examine the species specificity of BLyS, mouse splenic B cells were cultured in the presence of human BLyS (HuBLyS) and SAC. Recombinant BLyS induced in vitro proliferation of murine splenic B cells and bound to a cell-surface receptor on these cells. Immature surface Ig-negative B cell precursors isolated from mouse bone marrow did not proliferate in response to BLyS, nor did they bind the ligand (6).

To assess the in vivo activity of rBLyS, BALB/c mice (three mice per group) were injected intraperitoneally (i.p.) twice a day for 4 days with buffer only or with BLyS (2 mg/kg body weight). Upon treatment with BLyS, normal splenic architecture was altered by an expansion of the white pulp and an increase in nucleated cells within the red pulp (RP) (Fig. 4A). The B cell regions within the periarterial lymphatic sheaths (PAL) and the marginal zone were expanded but appeared to stain less intensely with the B cell marker CD45R (also known as B220). In addition, the T cell–dense regions surrounding the central arterial (CA) were also infiltrated by moderate numbers of CD45R+ cells. This suggests the white pulp changes were due to increased numbers of B cells. The densely packed cell population that frequently filled RP spaces did not stain with CD45R.

Figure 4

In vivo effects of BLyS administration in BALB/cAnNCR mice. (A) Splenic architecture of normal and BLyS-treated mice. The lower panels are sections taken from the same animals stained with a mAb to CD45R and developed with horseradish peroxidase–coupled rabbit anti-rat Ig (mouse adsorbed) and the substrate diaminobenzidine tetrahydrochloride (DAB) (34). CD45R-expressing cells appear brown. (B) Flow cytometric analyses of normal (left panel) and BLyS-treated (right panel) cells stained with PE-CD45R(B220) and FITC-Ly6D(ThB). Cells were obtained from the mouse spleens shown in Fig. 4A. (C) Serum IgM, IgG, and IgA levels in normal and BLyS-treated mice were quantitated using isotype-specific enzyme-linked immunosorbent assays.

Flow cytometric analyses of the spleens from BLyS-treated mice indicated that BLyS increased the proportion of CD45Rdull, Ly6Dbright (also known as ThB) B cells approximately 10-fold over that observed in control mice (Fig. 4B). This phenotype is rare among normal splenocytes but is characteristic of terminally differentiated plasma cell populations (9, 10). A potential consequence of increased B cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM, IgG, and IgA concentrations were compared between buffer- and BLyS-treated mice (Fig. 4C). BLyS administration resulted in five- and twofold increases in serum IgM and IgA, respectively. Circulating IgG concentrations did not increase after 4 days treatment with BLyS.

Here, we define BLyS as a member of the TNF superfamily that induces both in vivo and in vitro B cell proliferation and differentiation. BLyS is distinguished from other B cell growth and differentiation factors such as IL-2 (11), IL-4 (12, 13), IL-5 (14, 15), IL-6 (16, 17), IL-7 (18,19), IL-13 (20), IL-15 (21), CD40L (22, 23), or CD27L (CD70) (24, 25) by its monocyte-specific gene and protein expression pattern and its specific receptor distribution and biological activity on B lymphocytes. BLyS is likely involved in the exchange of signals between B cells and monocytes or their differentiated progeny. Although all B cells may use this mode of signaling, the restricted expression patterns of BLyS receptor and ligand suggest that BLyS may function as a regulator of T cell–independent responses in a manner analogous to that of CD40 and CD40L in T cell–dependent antigen activation (26,27). As such, BLyS, its receptor, or related antagonists may find medical utility in the treatment of B cell disorders associated with autoimmunity, neoplasia, or immunodeficiency syndromes.

  • * To whom correspondence should be addressed. E-mail: david_hilbert{at}hgsi.com

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