A designer hyper interleukin 11 (H11) is a biologically active cytokine

Abstract

Background: Interleukin 11 (IL-11) is a pleiotropic cytokine with anti-apoptotic, anti-inflammatory and hematopoietic potential. The IL-11 activity is determined by the expression of the IL-11R receptor alpha (IL-11Rα) and the signal transducing subunit β (gp130) on the cell membrane. A recombinant soluble form of the IL-11Rα (sIL-11Rα) in combination with IL-11 acts as an agonist on cells expressing the gp130 molecule. We constructed a designer cytokine Hyper IL-11 (H11), which is exclusively composed of naturally existing components. It contains the full length sIL-11Rα connected with the mature IL-11 protein using their natural sequences only. Such a construct has two major advantages: (i) its components are as close as possible to the natural forms of both proteins and (ii) it lacks an artificial linker what should avoid induction of antibody production.

Results: The H11 construct was generated, the protein was produced in a baculovirus expression system and was then purified by using ion exchange chromatography. The H11 protein displayed activity in three independent bioassays, (i) it induced acute phase proteins production in HepG2 cells expressing IL-11, IL-11Rα and gp130, (ii) it stimulated the proliferation of B9 cells (cells expressing IL-11Rα and gp130) and (iii) proliferation of Baf/3-gp130 cells (cells not expressing IL-11 and IL-11Rα but gp130). Moreover, the preliminary data indicated that H11 was functionally distinct from Hyper-IL-6, a molecule which utilizes the same homodimer of signal transducing receptor (gp130).

Conclusions: The biologically active H11 may be potentially useful for treatment of thrombocytopenia, infertility, multiple sclerosis, cardiovascular diseases or inflammatory disorders.

Figure 1

(A) Amino acid (AA) sequence of the fusion protein H11 and (B) Schematic representation of tertiary structure of the fusion complex. (A) The N-terminal signal peptide is indicated by thick underline. The sequences of C-terminus of IL-11Rα and N-terminus of IL-11, which form natural linker are boldface. The arrow indicates the fusion site. (B) The Ig-like region (D1 domain) and cytokine binding domains (D2 and D3) of IL-11Rα are indicated. The three receptor binding sites of IL-11 are shown. IL-11 binds the IL-11Rα though site I, while site II and III are occupied by two gp130 molecules.

Figure 2

Purification of H11-SDS-PAGE (A) and Western Blot (B) analysis. The proteins were visualized with Roti-Blue stain and for detection H11 the anti- IL-11Rα antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used. Lane 1- molecular weight marker PageRuler (Fermentas), lane 2-supernatant from infected High-Five BTI-TN-5B1-4 cells, 3-supernatant diluted with 20 mM 1.3 DAP (1:8), 4-eluted H11.

Figure 3

Rocket immunoelectrophoresis of supernatants from HepG2 cells. HepG2 cells were stimulated with recombinant H11 at various concentrations. After 48 h the collected medium was analyzed by rocket immunoelectrophoresis for presence of α₁-antitrypsin.

Figure 4

The proliferation assay of B9 cells. B9 cells (1 × 10⁴ cells per well) were tested for proliferation at various concentrations of (A) IL-11 and (B) H11. The medium without stimulator was used as the negative control. The proliferation of the cells was measured after 3 days using colorimetric assay based on MTT reagent.

Figure 5

The proliferation assay of Ba/F3-gp130 cells. Ba/F3-gp130 (2 × 10⁴ cells per well) cells were induced by H11 at various concentrations. The medium without stimulator was used as the negative control. The proliferation of the cells was measured after 3 days using colorimetric assay based on MTT reagent.

Figure 6

Phosphorylation of transcription factor STAT 3 analyzed by flow cytometry. Ba/F3 and Ba/F3-gp130 cells were stimulated with control medium (unstimulated), hIL-11 or H11 and phosphorylation of STAT3 was measured using anti- STAT3P antibody.

Figure 7

Effect of Hyper-IL-6 and H11 on the differentiation of cord blood-derived lineage-depleted CD34+ hematopoietic progenitor cells. Cells were seeded in X-Vivo 10 medium supplemented with rhSCF and rhIL-6 (control) and in the presence of Hyper-IL-6 or H11. After day 14 cells were harvested and analyzed by flow cytometry for the expression of CD15 (A), and CD235a (B).