Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro

Abstract

Granulomatous lymphadenitis is one of the pathognomonic lesions in post-weaning multisystemic wasting syndrome (PMWS)-affected pigs. This unique lesion has not been reported in direct association with viral infection in pigs. The objective of the present study was to evaluate whether porcine circovirus type 2 (PCV2) alone is able to induce functional modulation in porcine monocytic cells in vitro to elucidate its possible role in the development of granulomatous inflammation. It was found that the proliferation activity of blood monocytes (Mo) and monocyte-derived macrophages (MDM) was significantly enhanced by PCV2. During monocyte-macrophage differentiation, the PCV2 antigen-containing rate and formation of multinucleated giant cells (MGC) were significantly increased in MDM when compared to those in Mo. The MDM-derived MGC displayed a significantly higher PCV2 antigen-containing rate than did the mono-nucleated MDM. Supernatants from PCV2-inoculated MDM at 24 h post-inoculation induced an increased tendency of chemotactic activity for blood Mo. At the same inoculation time period, levels of mRNA expression of the monocytic chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1, also significantly increased in PCV2-inoculated MDM. The results suggest that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PMWS-affected pigs.

Figure 1.

Characterisation of porcine blood monocytes (Mo) and monocyte-derived macrophages (MDM) by flow cytometry following staining with anti-SWC1 (A) and anti-SWC9 (B) mAb. In (A) and (B), the filled-histograms represent the Mo collected on day 0, and the opened histograms represent the MDM after 3 days of incubation with 30% of pooled PCV1-, PCV2-, and PRRSV-free porcine plasma. The data of the positive rates of SWC1 and SWC9 (C) as well as MHC I and MHC II (D) are expressed as percentages and shown as mean ± SD of 6 pigs from three independent experiments in triplicate. *The difference between Mo and MDM is statistically significant (p < 0.05). (C) ■: SWC1, □: SWC9; (D) ■: MHC I, □: MHC II.

Figure 2.

Changes in total cell counts and cell survival rate of mock- and porcine circovirus type 2 (PCV2)-inoculated porcine monocytes (Mo) (A) and monocyte-derived macrophages (MDM) (B) over time as determined by trypan blue dye exclusion assay. Data are shown as mean ± SD of 6 pigs from three independent experiments in triplicate. *The difference between PCV2- and mock-inoculated groups at the same hour post-inoculation (HPI) is statistically significant (p < 0.05). ■: total cell counts of mock-inoculated, □: total cell counts of PCV2-inoculated, –■–: cell survival rate of mock-inoculated, –□–: cell survival rate of PCV2-inoculated.

Figure 3.

Porcine circovirus type 2 (PCV2)-induced cell fusion in blood monocyte-derived macrophages (MDM). Formation of multinucleated giant cells (MGC) (arrows) is compared between mock-inoculated MDM (A) and PCV2-inoculated MDM (B) as shown by co-labelling the nuclei and PCV2 antigen with Hochest stain and fluorescein isothiocyanate (FITC) by indirect immunofluorescence antibody staining, respectively. Note spontaneous cell fusion, mainly bi- and tri-nucleated, in mock-inoculated group (A, arrowheads) and significantly higher nuclear number in PCV2-inoculated group (B, arrows). (A color version of this figure is available at www.vetres.org.)

Figure 4.

The chemotactic effect of the supernatants from mock- and PCV2-inoculated monocyte-derived macrophages (MDM) collected at 24 h post-inoculation (HPI) on blood monocytes (Mo) as determined by a migration assay using Transwell^® plates. Data are expressed as (A) the total number of Mo attributed to the lower chambers shown as mean ± SD of three pigs of the Transwell^® plate and (B) dot plot of each of the three pigs used in the experiment by showing the total number of Mo attracted to the lower chamber of the Transwell^® plate. (A) □: medium control, ■: mock-inoculated, : PCV2-inoculated; (B) ●: medium control, ▽: mock-inoculated, ■: PCV2-inoculated.

Figure 5.

The mRNA expression levels of monocytic chemokines, MIP-1 and MCP-1, in mock- and PCV2-inoculated porcine monocyte-derived macrophages (MDM). The mRNA expression levels of MIP-1 and MCP-1 and the corresponding housekeeping genes G3PDH and β-actin were analyzed at 0 and 24 h post-inoculation (HPI) by RT-PCR and gel electrophoresis. The results of gel electrophoresis of MIP-1 (A-1) and MCP-1 (A-2) at 24 HPI and corresponding G3PDH and β-actin are displayed. The mRNA expression levels of MIP-1 (B-1) and MCP-1 (B-2) were further normalized using the geometric mean of housekeeping genes G3PDH and β-actin and the values are expressed as relative levels of mRNA expression and shown as mean ± SD of three pigs from two independent experiments. *The difference between PCV2- and mock-inoculated groups is statistically significant (p < 0.05). Mock: MDM inoculated with an equal volume of RPMI-C; PCV2: MDM inoculated with PCV2 at an m.o.i. of 1. ■: mock-inoculated, □: PCV2-inoculated.