Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin

Abstract

The Epstein-Barr virus-induced gene 3 (EBI3) is a novel soluble hematopoietin component related to the p40 subunit of interleukin 12 (IL-12). When EBI3 was expressed in cells, it accumulated in the endoplasmic reticulum and associated with the molecular chaperone calnexin, indicating that subsequent processing and secretion might be dependent on association with a second subunit. Coimmunoprecipitations from lysates and culture media of cells transfected with expression vectors for EBI3 and/or the p35 subunit of IL-12 now reveal a specific association of EBI3 with p35. Coexpression of EBI3 and p35 mutually facilitates their secretion. Most importantly, a large fraction of p35 in extracts of the trophoblast component of a human full-term normal placenta specifically coimmunoprecipitated with EBI3, indicating that EBI3 is in a heterodimer with p35, in vivo. Because EBI3 is expressed in EBV-transformed B lymphocytes, tonsil, spleen, and placental trophoblasts, the EBI3/p35 heterodimer is likely to be an important immunomodulator.

Figure 1

EBI3 is secreted in association with IL-12 p35 from cotransfected cells. (A) Immunoblots with affinity-purified rabbit EBI3 antibody detect EBI3 in p35Flag immunoprecipitates from cotransfected BJAB cell lysate (Upper) or cell supernatant (Lower). BJAB cells (10⁷ per transfection) were electroporated with 40 μg of pSG5 vector expressing EBI3 (9), EBI3Flag (9), p35Flag, or FlagLMP1 as indicated by a plus at the top of the figures. Twenty-four to 48 h posttransfection, cells or culture media were harvested and 1% digitonin cell extracts or cell culture media were immunoprecipitated with M2 anti-Flag monoclonal antibody. Fractions of the cell lysates or cell culture media obtained before immunoprecipitation (lanes 1–5) or the immunoprecipitates (lanes 6–10) were analyzed by 10% SDS/PAGE gel and immunoblotting. Molecular mass markers (in kilodaltons) are indicated on the left. In data not shown, immunoblots with M2 anti-Flag monoclonal antibodies or rabbit polyclonal IL-12 antisera indicated that about 20% of the total Flag-tagged proteins were immunoprecipitated from the cell. The efficiency of p35Flag immunoprecipitation from the culture media could not be determined because secreted p35Flag was only detected after immunoprecipitation. (B) EBI3 immunoblots detect EBI3 in anti-Flag immunoprecipitates from the culture media of COS7 cells coexpressing EBI3 with p35Flag, but not in that of COS7 cells coexpressing EBI3 with p40Flag. COS7 cells (4 × 10⁶ per transfection) were electroporated with 12 μg of pSG5 vector expressing EBI3, p35Flag, or p40Flag as indicated, and 3.5 days posttransfection, cell lysates and culture media were coimmunoprecipitated as described in A, except that cells were lysed in 0.5% NP40. Immunoblot using M2 anti-Flag monoclonal antibodies or rabbit polyclonal IL-12 antisera indicated that about 20% of the cell-associated Flag-tagged proteins and most of the secreted Flag-tagged proteins were immunoprecipitated (not shown). (C) Immunoblot analysis of coimmunoprecipitation experiments performed from the lysates of transfected COS7 cells. COS7 cells (4 × 10⁶ per transfection) were transfected with 20 μg of pSG5 vector expressing the proteins indicated at the top of the figures and lysed 48 h posttransfection in 0.5% Nonidet P-40 lysis buffer. Lysates were immunoprecipitated with M2 anti-Flag antibody (Left, lanes 6–10) or EBI3 affinity-purified rabbit polyclonal antibodies (Right, lanes 5–8). A fraction of the lysate (10% input) and immunoprecipitates were analyzed by immunoblotting using IL-12 antisera (Upper) or EBI3 anti-sera (Lower). IL-12 p35 migrates at the level of the IL12 blot lettering and just above the 31-kDa marker.

Figure 2

EBI3 and IL-12p35 can associate in solution. COS7 cells (4 × 10⁶ per transfection) were electroporated with 12 μg of pSG5 plasmids expressing the proteins indicated by a plus at the top of the figure, and culture media were immunoprecipitated with M2 anti-Flag antibody. A fraction of the culture media (lanes 1–4) or immunoprecipitates (lanes 5–8) was analyzed by immunoblotting with affinity-purified EBI3 antibodies. Lanes 3 and 7 correspond to media from cells cotransfected with EBI3 and p35Flag expression vectors, whereas lanes 4 and 8 correspond to media from cells that were independently transfected with EBI3 or p35Flag expression vectors and then cocultured. These later cells were incubated at 37°C for 15 h after transfection, washed twice with PBS, trypsinized, and then cocultivated for 72 h before harvest. Immunoblot with M2 anti-Flag antibody indicated that most Flag-tagged proteins were precipitated (data not shown). As a control for the absence of cotransfected cells, EBI3 was not detected in an anti-Flag immunoprecipitate from the lysate of cocultures of p35Flag transfected cells and EBI3 transfected cells (data not shown).

Figure 3

Effect of EBI3 coexpression on p35 secretion. (A) BJAB cells (10⁷ cells per transfection) were transfected with 30 μg of pSG5 vector control or pSG5 vector expressing EBI3Flag, p35Flag, or p40Flag. Approximately 24 h posttransfection, cells were labeled with [³⁵S]Met/Cys for 18 h, and 0.5% Nonidet P-40 extracts (cell lysate, lanes 1–4) or culture media (cell sup, lanes 5–8) were immunoprecipitated with M2 anti-Flag antibody. Immunoprecipitates were separated on 10% SDS/polyacrylamide gel and analyzed by autoradiography. The positions of EBI3Flag, p35Flag, and p40Flag are indicated by a horizontal line, filled circle, or open circle, respectively. (B) BJAB cells (10⁷ cells) were transfected with the indicated amount (in micrograms) of pSG5 vector expressing p35Flag or EBI3. The total amount of transfected DNA was maintained constant by addition of pSG5 vector. Approximately 20 h posttransfection, cells were labeled with [³⁵S]Met/Cys and processed as in A. Lanes 1–3 are immunoprecipitates from cell lysates, and lanes 4–6 are immunoprecipitates from culture media.

Figure 4

Effect of p35 coexpression on EBI3 secretion. COS7 cells (4 × 10⁶ per transfection) were transfected with the indicated amount (in micrograms) of pSG5 vector expressing EBI3 or p35Flag. Total transfected DNA was maintained constant by the addition of pSG5 vector. After 20 h, cells were harvested and lysed in SDS buffer. Culture media were immunoprecipitated with EBI3 antisera. Lysates (A) and immunoprecipitates from culture media (B) were analyzed by SDS/PAGE and immunoblotting with EBI3 antisera. The position of EBI3 is indicated by the arrow.

Figure 5

EBI3 and IL-12p35 coprecipitate from placenta extract. Immunoblots with rabbit affinity-purified EBI3 antibodies (Upper) or IL-12 antisera (Lower) of immunoprecipitates from extracts of human placenta. Trophoblast villi were dissected from a normal, full-term human placenta, and 0.5% Nonidet P-40 extracts from the equivalent of about 10⁸ cells were immunoprecipitated with affinity-purified rabbit EBI3 antibodies (EBI3) or preimmune rabbit sera (NRS). Immunoprecipitates (100%) or cell lysate obtained before immunoprecipitation (1%) were analyzed by SDS/PAGE on a 10% gel. No signal was detected in immunoprecipitates with irrelevant rabbit polyclonal antibodies (data not shown). We cannot be certain that the band oberved in the lysate (Lower, lane 1), which is equivalent in mobility to p35 in the EBI3 immunoprecipitate (Lower, lane 3) is entirely p35, because multiple bands were detected in the cell lysate.