Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility

Abstract

Porcine monomyeloid cell lines were established following transfection of primary porcine alveolar macrophage cultures with plasmid pSV3neo, carrying genes for neomycin resistance and SV40 large T antigen. The parental clone 3D4 exhibited a relatively rapid doubling time (25.5 h), high plating efficiency and mixed phenotype with respect to growth on a solid support. Single cell cloning of the 3D4 parent resulted in establishment of several cell lines; three of them designated 3D4/2, 3D4/21 and 3D4/31 were selected for further characterization. All three clones supported the replication of vesicular stomatitis virus (VSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), swine poxvirus, African swine fever virus (ASFV), herpes simplex virus (HSV), parainfluenza virus, bovine adenovirus (BAV), vaccinia virus (VV), and porcine adenovirus (PAV). Under the conditions tested the cells did not support replication of porcine reproductive and respiratory syndrome virus (PRRSV). The swine myeloid character was confirmed for all three clones by fluorescence activated cell scanning (FACS) analysis using monoclonal antibodies 74-22-15 and DH59B, which recognize the pan-myeloid antigen cluster SWC3a. A subpopulation of each cell line was positive for nonspecific esterase activity and phagocytic activity to varying degrees depending on the media formulation. Cells from all three lines exhibited anchorage dependent growth when maintained in RPMI 1640 medium supplemented with 5-15% fetal bovine serum (FBS) and nonessential amino acids. Propagation in commercially formulated serum free media resulted in colony formation and growth in suspension. The addition of dimethyl sulfoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) to serum free media restored cell attachment. DMSO was also able to induce expression of CD14 monocyte marker in the 3D4/31 cell line maintained in FBS containing medium, as determined by FACS with monoclonal antibody CAM36A. Supplementation of RPMI medium with 10% porcine serum upregulated the expression of CD14 and induced expression of porcine macrophage markers recognized by antibodies 2B10 and 2G6 (Vet. Immunol. Immunopathol. 74 (2000) 163) in all three cell lines. The porcine myelomonocytic cell lines obtained may have a wide variety of applications in porcine virology and immunology.

Fig. 1

Growth curve of the continuous porcine myelomonocytic single cell clones 3D4/2 (♦), 3D4/21 (■) and 3D4/31 (▴) in 15% FBS/RPMI. Cells were seeded in six-well plates at approximately 2×10⁵ cells/well in 15% FBS/RPMI, nonessential amino acids (1:100) and gentamycin (5 μg/ml). Three wells per clone were trypsinized at 22, 28, 48, 72, 96, 116 and 142 h post seeding, and the cells from each well were counted The obtained average cell count for each clone at each time point was plotted against the time.

Fig. 2

Immunofluorescent staining detected by flow cytometry of the clone 3D4/31 incubated overnight in either DMSO/FCS/RPMI (DMSO) or 10% porcine serum/RPMI (PorS), and labeled with antibody 2B10 (A) or antibody 2G6 (B). Cell control fluorescence intensity was in the range illustrated in panel PorS (no fill peak).

Fig. 3

FACScan analysis of clones 3D4/2, 3D4/21 and 3D4/31 for immune cell surface markers. Cells were incubated in 15% FBS/RPMI (FBS), 1 μM PMA/FBS/RPMI (PMA), 1% DMSO/FCS/RPMI (DMSO), 10% porcine serum/RPMI (PorS), and macrophage-SFM medium (SFM), prior to labeling with the following antibodies: 74-22-15 (white column), DH59B (right leaning dashed column), CAM36A (black), 2B10 (dotted), 2G6 (left leaning dashed column). Primary antibodies were incubated with FITC conjugated rabbit anti-mouse F(ab′)₂ secondary antibody, and analyzed using a Becton Dickinson FACScan.

Fig. 4

Virus susceptibility of the continuous porcine myelomonocytic cell lines 3D4/2, 3D4/21 and 3D4/31 in 7.5% FBS/RPMI, compare to the susceptibility of standard assay cells: 3D4/2 (dotted column), 3D4/21 (white), 3D4/31 (dashed), standard assay cells (black column). Panel A illustrates results when cells were seeded in 96-well plates and incubated overnight in RPMI containing 15% FBS. The maintenance medium was then replaced with 10-fold dilutions of various virus stocks made in RPMI and incubated with the cells for 3 h at 37 °C, 5% CO₂. After the adsorption period, the virus inoculum was removed and cells were washed once with RPMI and overlayed with their respective maintenance medium containing 7.5% of FBS. Cells were incubated for 5 days at 37 °C, 5% CO₂ and then either fixed for immunostaining (ASFV, CSFV) or evaluated for the degree of cytopathic effect (CPE) (SVDV, VSV, PRV). In the case of cells infected with SwPV, the infected cell supernatant was titrated using a susceptible cell line (ST). Panel B illustrates results when viruses were successively passed twice in each of the three cell lines and the supernatants from the second passage were then titrated (10-fold dilutions) on previously determined, susceptible cell lines along with the original virus inoculum. Infection of the IPAM cell clones was carried out by incubating a 0.5 ml cell suspension (2×10⁵ cells/ml) made in RPMI containing 7.5% FBS with 0.5 ml of virus inoculum in a 12-well plate for 5 days at 37 °C, 5% CO₂. Prior to preparing the cell suspension, the IPAM cell clones were maintained in the above medium containing 15% FBS.