Abstract
ABSTRACTThis study investigated the heterogeneity and plasticity of porcine alveolar macrophages (PAM) and pulmonary interstitial macrophages (IM) isolated from healthy pigs, including phenotype, function and gene expression. Dynamic changes of nitric oxide (NO) levels secreted by PAM and IM with stimulation of different doses of lipopolysaccharide (LPS) were investigated by Griess method, and the viability of the PAM and IM cells was investigated by MTT assay. Flow cytometry, fluorescence quantitative PCR and ELISA techniques were used to measure cell phenotype, gene expression and cytokine secretion, respectively. The PAM and IM cells in normal healthy pigs showed heterogeneity with 95.42±1.51% and 31.99±5.84% of CD163+ macrophage, respectively. The NO level in IM was significantly higher versus PAM after LPS treatment. Consistently, the ratio of Arg I/iNOS in IM was much lower than that in PAM, suggesting that the PAM belong to M2 macrophages and the IM belong to M1 macrophages. The PAM and IM cells in normal healthy pigs also showed plasticity. The Arg I/iNOS ratio and TIMP1/MMP12 ratio were significantly decreased in LPS- or LPS+IFNγ-treated PAM and IM, suggesting that cells were polarized towards M1 macrophages under LPS or LPS+IFNγ stimulation. On the contrary, IL-4 and IL-13 stimulation on PAM and IM lead to M2 polarization. A similar result was found in IL-1β gene expression and TNFα secretion. In conclusion, porcine macrophages have shown heterogeneity and plasticity on polarization under the stimulation of LPS, IFNγ, IL-4 and IL-13.
Highlights
Macrophages form a front line of the host defense system that can express diverse activities depending on the stimuli encountered
The results showed that CD163+rate of interstitial macrophage (IM) cells was significantly decreased compared to that of porcine alveolar macrophage (PAM) cells (P
Dynamic changes of nitric oxide (NO) level secreted by PAM and IM stimulated with different doses of LPS PAM and IM cells isolated from healthy pigs were stimulated with different doses of LPS (1 pg/ml, 10 pg/ml, 100 pg/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml and 1 μg/ml) in vitro and NO level and cell viability were examined (Fig. 2)
Summary
Macrophages form a front line of the host defense system that can express diverse activities depending on the stimuli encountered. Many of these activities appear to be mutually antagonistic in nature, such as pro-inflammatory versus anti-inflammatory. In microenvironments dominated by lipopolysaccharide (LPS) or IFNγ, macrophages undergo M1 polarization, which leads to the increase of microbicidal capacity and inflammatory injury. The in vivo function of less mature forms of porcine monocytes, which are believed to play an important role as precursors of inflammatory macrophages in mice and humans, has not been reported and remains completely unknown in pigs (Ondrackova et al, 2010). The aim of this study was to discover the heterogeneity and plasticity of porcine alveolar macrophage (PAM) and pulmonary interstitial macrophage (IM) cells in normal healthy pigs in vitro, which will be helpful for the understanding of pathologic mechanisms and the prevention of swine infectious disease
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