Genetically engineered macrophages expressing IFN-gamma restore alveolar immune function in scid mice

Abstract

Reversal of immunodeficiency in the lung by gene therapy is limited in part by the difficulty of transfecting lung cells in vivo. Many options exist for successfully transfecting cells in vitro, but they are not easily adapted to the in vivo condition. To overcome this limitation, we transduced macrophages in vitro with the murine IFN-gamma (mIFN-gamma) gene and intratracheally delivered the macrophages to express mIFN-gamma in vivo. A recombinant retroviral vector pSF91 system was modified to encode mIFN-gamma and enhanced green fluorescent protein (EGFP). A murine macrophage cell line J774A.1 transduced with the retroviral supernatant increased secretion from undetectable levels to 131.6 +/- 4.2 microg/ml mIFN-gamma at 24 h in vitro. The mIFN-gamma-producing macrophages were intratracheally instilled into mechanically ventilated scid mice. mIFN-gamma levels in the bronchoalveolar lavage increased from undetectable levels at baseline to 158.8 +/- 5.1 pg/ml at 48 h (P < 0.001). Analysis of the lavaged cells for EGFP expression revealed that EGFP expression was directly proportional to the number of transduced macrophages instilled into the lung. Immune function was partially restored in the alveolar spaces of scid mice with evidence of enhanced MHC class II antigen expression and increased phagocytosis (P < 0.05). Tumor necrosis factor alpha was increased from undetectable at baseline to 103.5 +/- 11.4 pg/ml. In contrast, i.p. administration of the engineered macrophages did not enhance IFN-gamma levels in the lung. Our study suggests airway delivery of genetically engineered macrophages expressing mIFN-gamma gene can partially restore significant immune activity in the lungs of immunodeficient mice.

Figure 1

Construction of mIFN-γ-producing vector on MESV backbone. (a) Diagram of MESV retroviral vector for producing IFN-γ. From 5′-end: long terminal repeat (LTR), multiple clone sites (MCS) with the mIFN-γ insert, internal ribosome entry site (IRES), EGFP, and 3′-end LTR. The insertion of mIFN-γ was done with a PCR method at the EcoRI and SalI sites. The arrow indicates the transcription start site. The transcript contains IFN-γ, the vector's splice donor and splice acceptor sites along with the EGFP driven by IRES promoter. (b) Agarose gel analysis for the vector construct pSF91-EGFP-mIFN-γ. The positive E. coli HB101 clones expressing mIFN-γ demonstrated a band at 470 bp of EcoRI–SalI fragment corresponding to the mIFN-γ gene. Lanes: C, clone 1 (no digestion); V1, V2, vector; 1–4, clones of mIFN-γ constructs. (c) Reverse transcription PCR of mIFN-γ. (Left) mIFN-γ bands assessed with mIFN-γ primers. Left lane, vector control; right lane, engineered macrophages. (Right) Actin bands assessed with actin primers. Left lane, vector control; right lane, engineered macrophages. (d) Western blot of mIFN-γ in cultured cells and BAL cells assessed by monoclonal antibodies against mIFN-γ (16 kDa). Lanes: V, vector control; C, cultured engineered macrophages; B, BAL cells of scid mice receiving engineered macrophages.

Figure 2

J774A.1 macrophages expressing mIFN-γ identified by EGFP in vitro and in vivo. (Top) A representative (clone 10) of selected EGFP-positive J774A.1 clones after FACS sorting and cloning (over 90% cells are EGFP-positive), viewed with phase-contrast (A) and fluorescent (B) microscopy. (×800.) (Middle) Detection of J774A.1 macrophages expressing EGFP in scid mouse BAL cells 48 h after instillation by using phase-contrast (C) and fluorescent (D) microscopy. (×400.) Note that residential AMs have a different morphology, and are smaller compared with J774A.1 cells. (Bottom) EGFP-expressing cells in the lung 24 h after instillation, viewed by phase-contrast (E) and fluorescent (F) microscopy. (×400.) A typical J774A.1 cell is shown with an arrowhead in D and F.

Figure 3

Expression of mIFN-γ protein and EGFP in vitro and in vivo. (a) Time-dependent expression of mIFN-γ in cell culture from engineered J774A.1 macrophages determined by ELISA. (b) In vivo mIFN-γ expression in BALB/c scid mice including serum and BAL from normal controls or from mice 48 h after intratracheal instillation of J774A.1 macrophages with vector control or mIFN-γ gene (∗, P < 0.001, compared with control). (c and d) Effect of concentration of airway-delivered J774A.1 macrophages expressing mIFN-γ on number of EGFP-expressing BAL cells assessed by FACS (c, 24 h) and on mIFN-γ levels in BAL assessed by ELISA (d, 24 h). Expression of mIFN-γ correlates with the number of J774A.1 macrophages expressing mIFN-γ (*, P < 0.05).

Figure 4

Time-dependent in vivo expression of mIFN-γ in BAL after intratracheal instillation of 10⁶ mIFN-γ-expressing macrophages into scid mice. IFN-γ expression peaked at the first week and eventually tapered, but was detectable for a month.

Figure 5

Expression of MHC class II antigen (I–A/I–E) in BAL cells of scid mice receiving J774A.1 expressing mIFN-γ. (a) Expression of I–A/I–E class II antigen from 1–14 days, assessed by PE-labeled anti-IA/I-E antibodies. (∗, P < 0.05, compared with control.) (b) Fluorescent microscopy of BAL cells expressing class II antigen (I-A/I-E) 48 h after intratracheal instillation of the engineered J774A.1 macrophages expressing mIFN-γ. (A and B) Cells with vector control showed by phase-contrast (A) and PE fluorescent (B) microscopy. (×400.) (C and D) Cells expressing mIFN-γ as shown by phase-contrast (C) and PE fluorescent (D) microscopy. (×400.)

Figure 6

Phagocytosis by BAL cells after intratracheal instillation of the engineered J774A.1 macrophages expressing mIFN-γ into scid mice. (a) Phagocytic function of BAL cells determined by Texas red-labeled E. coli particles from BALB/c scid mice 1–14 days after receiving J774A.1 macrophages expressing mIFN-γ (∗, P < 0.05, compared with control). (b) Phase-contrast and fluorescent microscopy of BAL cells phagocytizing Texas red-labeled E. coli particles. (×400.) (Upper) BAL cells from mice receiving J774A.1 containing vector control shown by phase-contrast (A) and fluorescent (B) microscopy and merged images (C). (Lower) BAL cells from mice receiving J774A.1 expressing mIFN-γ as shown by phase-contrast (D) and fluorescent (E) microscopy and merged images (F).

Figure 7

Cytokine expression in BAL from scid mice following intratracheal instillation of J774A.1 expressing mIFN-γ. (a) Cytokine expression in BAL 48 h after instillation of macrophages expressing mIFN-γ and vector control (∗, P < 0.01, compared with control). (b) MHC class II expression by BAL cells 24 h after intratracheal instillation of J774A.1 expressing mIFN-γ into scid mice. In the right-most column the BAL cells were stimulated with E. coli stereotype 026:B6 lipopolysaccharide (LPS) at 10 μg/ml for a further 24 h (∗, P < 0.05, compared with control).

Figure 8

Comparison of cytokine production between airway and i.p. delivery of J774A.1 expressing mIFN-γ. (a) mIFN-γ production in BAL from scid mice (24 h) receiving J774A.1 macrophages expressing mIFN-γ by airway (IT) and i.p. delivery (∗, P < 0.01, compared with control). (b) TNF-α production in BAL from scid mice (24 h) receiving J774A.1 macrophages expressing mIFN-γ by airway and i.p. delivery (∗, P < 0.01, compared with control).