doi:10.1016/S0008-8749(03)00129-1 
Copyright © 2003 Elsevier Science (USA). All rights reserved.
Placental protein 14 regulates selective B cell responses
Einat Yaniv, Zipora Borovsky, Galit Mishan-Eisenberg and Jacob Rachmilewitz
, 
Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel
Received 13 February 2003;
accepted 8 May 2003. ;
Available online 11 June 2003.
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Abstract
Placental protein 14 (PP14) is a glycoprotein of the lipocalin family that acts as a negative regulator in T cell receptor-mediated activation. In this study, we investigated PP14s potential role in regulating B cell activation. While PP14-inhibited B cell proliferation, IgM secretion and the surface expression of MHC class II, the expression of other surface molecules, such as CD69 and CD86, were unaffected. These observed effects were independent of the anti-IgM concentration used for stimulation, regardless of the presence of either T cells or IL-4, and persisted when B cells were stimulated by stimuli, which circumvent early events during B cell Ag receptor (BCR) activation, namely, protein kinase C activators in combination with Ca2+ ionophore. Interestingly, we demonstrated that PP14s inhibitory characteristics are reminiscence of that achieved by independent ligation of CD19 using anti-CD19 mAb. Together with our previously reported effects on T cells, these findings identify PP14 as a soluble regulatory factor capable of interacting with both T and B cells in a carbohydrate-dependent manner and as a result it can affect both cellular and humoral immune responses.
Author Keywords: Human; B lymphocytes; Cellular activation; PP14; CD19
Fig. 1. PP14 inhibits B cell proliferation induced by soluble anti-IgM or PMA and ionomycin, but not by solid-phase anti-IgM. Purified B cells were stimulated with increasing concentrations of either soluble (A) or solid-phase (B) anti-IgM mAbs, or by PMA plus ionomycin (C), in the presence of either AF (25% or 50%; v/v), or PP14·Fcγ1 (100 μg/ml, which corresponds to the estimated PP14 concentration in 50% AF). Proliferation was determined by [3H]thymidine uptake during the last 16 h of a 72 h assay. Values show the mean of triplicate samples. Each experiment is a representative of four similar experiments. Similar results were also obtained with soluble F(ab)2 fragment of anti-IgM used instead of the soluble anti-IgM Ab. A statistical significant decrease (P<0.01, using a Student’s t test) in B cell proliferation was observed for all cultures treated with either AF or PP14·Fcγ1.
Fig. 2. PP14 inhibits anti-IgM- and PMA and ionomycin-induced MHC II expression, but not CD69 and CD86 expression. Selective inhibition of B cell activation markers by PP14. Flow cytometric analysis of B cells stimulated with anti-IgM mAbs (Left panels), or by PMA plus ionomycin (Right panels) and stained with anti-MHC class II (A), anti-CD69 (B), and anti-CD86 (C). The results shown are representative of four different experiments.
Fig. 3. Inhibition of IgM secretion by PP14. Purified B cells were activated with PMA plus ionomycin in the absence or presence of PP14·Fc
γ1. After 8 days, the level of IgM in the medium was determined by ELISA. Data represent the mean of triplicate samples. Comparable results were obtained in three separate experiments. A statistical significant decrease (
P<0.01, using a Student’s
t test) in IgM release was observed for cultures treated with PP14·Fc
γ1.
Fig. 4. PP14 binds to B cell surface in a carbohydrate-dependent manner. Purified B cells were incubated with either PP14·Fc
1 or CD99·Fc
γ1 for 30 min at 37 °C. For purposes of competitive inhibition, cells were pre-incubated with either AF (50% v/v) or asialofetuin (1 mg/ml), for 20 min at 37 °C prior to the addition of PP14·Fc
γ1. Cells were prepared and labeled as described in Materials and methods, and 2 × 10
4 cells were analyzed by flow cytometry to detect bound protein. Comparable results were obtained in three separate experiments.
Fig. 5. Inhibition of B cell activation by anti-CD19 mAb. Purified B cells were stimulated by anti-IgM mAb (A and C), or with PMA plus ionomycin (B and D) alone, or in the presence of anti-CD19 (1 μg/ml), and the proliferative response, IgM secretion and CD69 expression was monitored as described in
Fig. 1,
Fig. 3 and
Fig. 2, respectively. Each experiment was confirmed in three independent experiments. A statistical significant decrease (
P<0.01, using a Student’s
t test) in B cell response was observed for cells treated with anti-CD19 mAb.
Abstract
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