Inflammation plays an essential role in respiratory defense mechanism . In porcine pleuropneumonia, multiple components and virulence factors of A. pleuropneumoniae are suggested to contribute to the production of proinflammatory cytokines . Baarsch et al. demonstrated alveolar lavage cells from pigs endotracheally inoculated with a low-virulence strain of A. pleuropneumoniae serotype 1, i.e., lacking cytotoxic and hemolytic activities, showed similar mRNA level of TNF, IL-1 or IL-8 compared to buffer treated pigs . Moreover, heat-treatment of crude A. pleuropneumoniae bacterial culture supernatant attenuated its ability to induce TNF-α and IL-1 expression in porcine alveolar macrophages (PAMs), suggesting heat-labile components were involved in such stimulation . In this study, we present direct evidence demonstrating a single species of exotoxin ApxI, derived from A. pleuropneumoniae serotype 10, induces the expression and production of proinflammatory cytokines IL-1β, IL-8 and TNF-α in PAMs.
Also, we found that, at low concentration such as 0.5 or 1 CU/mL, ApxI did not cause significant cell death within 4 h, and > 68% of PAMs survived at 12 h of stimulation (data not shown). Since alveolar macrophages are one of the first line defense cells in lungs, at the early stage of A. pleuropneumoniae infection, PAMs may be a source of inflammatory mediators contributing to innate immunity. Nevertheless, PAMs may not be the only source of proinflammatory cytokines during A. pleuropneumoniae infection. For instance, Baarsch et al. demonstrated expression of cytokine IL-6 in the lung tissue of pigs inoculated with live A. pleuropneumoniae bacteria . However, fibroblasts and epithelial cells, but not alveolar macrophages, might be the source of IL-6 . This is consistent with our findings showing PAMs did not express IL-6 upon ApxI stimulation. Therefore, whether ApxI stimulates inflammatory cytokine IL-1β, IL-8 or TNF-α in other cell types remains to be clarified.
Overproduction of proinflammatory mediators in lungs can harm animals . Based on our findings in this study, it is tempting to hypothesize, in addition to the hemolytic and cytolytic effects of ApxI, the toxin may exacerbate lung damages caused by A. pleuropneumoniae through the induction of proinflammatory cytokines. However, to avoid overemphasizing the effect of ApxI, it should be noted that the cell wall component of Gram negative bacteria, i.e., LPS, induces plethora of inflammatory and immunological effects in various cell types , such as TNF, IL-1 and IL-8 production by porcine macrophages [35–37]. Moreover, Ramjeet et al. had demonstrated that the cytotoxicity of ApxI and ApxII was enhanced through interaction with LPS . In our study, we found that in the absence of PMB, ApxI preparation induced significantly higher levels of proinflammatory cytokine expression as compared to treatment of ApxI in the presence of PMB (Figure 1A). These findings indicate both LPS and ApxI are cytokine inducing factors. However, whether LPS contributes to a synergistic or additive effect on ApxI-induced cytokine expression requires further investigation.
Mitogen-activated protein kinases (MAPKs) are important signaling molecules involved in the expression of inflammatory mediators in various types of cells [21–25]. Upon MAPK activation, down-stream targets including transcription factors are phosphorylated and activated that in turn bind to the promoter region of target genes . Cooperative involvement of multiple transcription factors is a common theme in the gene regulation of single cytokine. It has been demonstrated activating protein-1 (AP-1; consisting of heterodimers of c-Jun, activating transcription factor-2 (ATF-2), c-Fos, and Jun dimerization protein (JDP)), nuclear factor kappa B (NF-κB), and CAAT/enhancer-binding protein (C/EBP) participate in transcriptional control of IL-8 gene . Similarly, the expression of TNF-α gene is also regulated by C/EBP, NF-κB, and AP-1 (c-Jun/ATF-2) . In addition, it is well-established MAPKs may have overlapping substrate specificity on transcription factors . For example, c-Jun is phosphorylated and activated specifically by JNK, while ATF-2 can be phosphorylated by both JNK and p38 MAPKs [33, 40, 41]. Nevertheless, the involvement of MAPKs in RTX-induced cytokine gene expression has not been identified.
In the present study, we showed both p38 and JNK were substantially activated after ApxI stimulation, and these two signaling molecules played differential regulatory roles in cytokine gene expression. Our observations that p38 inhibitor (SB203580) attenuated the expression of IL-1β, IL-8 and TNF-α, while JNK inhibitor (SP600125) attenuated the expression only on TNF-α gene, suggest p38 plays a more important role in ApxI-induced IL-1β, IL-8 and TNF-α expression. These findings are similar to previous studies showing SB203580 inhibited gene expression of IL-1β, IL-8 and TNF-α [21, 23, 25], while SP600125 inhibited gene expression of TNF-α, but not IL-1β or IL-8 . However, the question of which transcription factors are involved in ApxI-induced cytokine expression and the regulatory role of MAPKs on these factors remain to be determined.
CD18 of β2 integrins has been proposed as a receptor for ApxIII-mediated leukolysis . In this study, the observations that blocking of CD18 on PAMs resulted in attenuation of ApxI-induced p38 and JNK activation and subsequent cytokine gene expression suggest ApxI-β2 integrin interaction contributes to these events. Nevertheless, current knowledge of signaling pathways underlying β2 integrin-induced MAPK activation is very limited. Studies by Hsuan et al. indicated M. haemolytica Lkt-induced intracellular [Ca2+] ([Ca2+]i) elevation in bovine alveolar macrophages is mediated through a G-protein-coupled mechanism , and [Ca2+]i elevation is crucial for proinflammatory cytokine gene expression . Consequently, a study by Jeyaseelan et al. demonstrated Lkt-induced [Ca2+]i elevation depends on LFA-1 . It has been reported MAPKs are activated by a small G-protein Ras downstream G-protein-coupled receptor (GPCR) signaling pathway [20, 43]. The question of whether ApxI-induced MAPK activation also occurs through a calcium-dependent, G-protein-coupled mechanism leading to cytokine gene expression requires further investigation.
In summary, this study provides evidence demonstrating ApxI is able to induce the expression of proinflammatory cytokines IL-1β, IL-8 and TNF-α in porcine alveolar macrophages. This is the first report to demonstrate p38 and JNK play differential regulatory roles in ApxI-induced cytokine gene expression through a CD18-dependent pathway.