Brain-heart infusion (BHI) was from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). The nicotinamide adenine dinucleotide (NAD), polymyxin B, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), trypan blue, and anti-β-actin antibody were from Sigma Aldrich (Saint Louis, MO, USA). The p38 inhibitor SB203580 and JNK inhibitor SP600125 were from Calbiochem (Darmstadt, Germany). Antibody specific to active, phosphorylated p38 or JNK was from Promega (Madison, WI, USA). A blocking antibody specific to porcine CD18 was purchased from SeroTec (Kidlington, Oxfordshire, United Kingdom).

Porcine alveolar macrophages (PAMs) were obtained from 3- to 6- week old healthy piglets through lavage and stored in liquid nitrogen using previously described procedures 27 . Piglets were euthanized according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing University. For the experiments, PAMs were thawed, resuspended in culture medium RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) and seeded into cell culture plates as indicated below.

Actinobacillus pleuropneumoniae serotype 10 (strain 13039) was a kind gift from the Animal Health Research Institute, Council of Agriculture, Republic of China. It secretes only ApxI but not ApxII and ApxIII 28 . Preparation of the ApxI was performed according to the procedures described previously 27 . Briefly, colonies of A. pleuropneumoniae serotype 10 on BHI agar plates containing 10 μg/mL NAD were transferred to BHI broth supplemented with 10 μg/mL NAD and cultured at 37°C for 5 h. The BHI broth was then replaced by RPMI-1640 supplemented with 2% FBS and 10 mM CaCl₂ and cultivated for an additional 2 h. Subsequently, the bacterial culture supernatant was collected by centrifugation at 16 000 × g for 10 min at 4°C followed by passage through a filter with pore-size of 0.45 μm. The filtered preparation containing native exotoxin ApxI was aliquoted and stored at -70°C for further experiments. The cytotoxic activity of ApxI was determined using an XTT assay as described previously 29 . One cytotoxic unit (CU) of ApxI was defined as the quantity of toxin causing a 50% reduction in mitochondrial activity of 2 × 10⁵ PAMs.

Limulus amebocyte lysate test (Cambrex Bio Science, Walkersville, MD, USA) was performed according to the manufacturer's instructions. It was found that the preparation containing 1 CU/mL ApxI had a level of 307 endotoxin units (EU)/mL. To minimize the effect of contaminating LPS in the exotoxin preparation, polymyxin B was added to a final concentration of 10 μg/mL throughout the study except where indicated otherwise.

PAMs were seeded to 35-mm tissue culture plates at a density of 2 × 10⁶ cells/plate in culture medium and incubated at 37°C in 5% CO₂ overnight. Cells were washed once and replenished with low serum medium (LSM; RPMI-1640 supplemented with 1% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) containing 0-2 CU/mL of ApxI and incubated for 0-12 h as indicated elsewhere. To abolish the bioactivity of ApxI, exotoxin was incubated at 98°C for 1 h or pre-incubated with 20 μg/mL antiserum raised against a recombinant subunit ApxI protein for 30 min at 37°C prior to applying to PAMs in the indicated experiments 27 . In experiments examining the roles of mitogen-activated protein kinases (MAPKs) and β2 integrins on ApxI-induced cytokine expression, PAMs were incubated with LSM containing 10 μM p38 inhibitor SB203580, 10 μM JNK inhibitor SP600125, or 5-10 μg/mL anti-porcine CD18 antibody for 1 h prior to stimulation with 0.5 CU/mL of ApxI.

The levels of cytokine mRNA in PAMs were evaluated by RT-qPCR at 2 h post ApxI stimulation as described above. Total RNA of PAMs was extracted using High Pure RNA Isolation Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. cDNA synthesis and RT-qPCR analysis were performed as follows.

To quantify the protein levels of cytokines, 2 × 10⁶ PAMs were stimulated with 1 CU/mL of ApxI for 4 h. In the inhibitor experiments, 1 × 10⁶ PAMs were incubated with 0.5 CU/mL ApxI for 8 h. After treatments, culture supernatants were collected following centrifugation at 700 × g for 10 min. The levels of cytokines IL-1β, IL-6, IL-8, and TNF-α in the culture supernatants were determined by quantitative DuoSet^® ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

The survival rate of PAMs was assessed using a trypan blue exclusion test 32 . Briefly, after ApxI treatment, PAMs were collected by trypsinization, stained with 0.1% trypan blue, and observed using a microscope. Cells stained blue were scored as non-viable. At least 500 cells were counted for each treatment of at least triplicate determinations. Percent of cell survival was calculated as follow: 100 × [1 - (% cell death at each time point - % cell death at 0 h)].

In experiments examining MAPK activation, 2 × 10⁶ cells PAMs in 35-mm tissue culture plates were stimulated with 0.5 CU/mL ApxI for 1 h. Thereafter, cells were washed once with ice-cold phosphate buffered saline (PBS) and lysed in a lysis buffer (1% Triton X-100, 20 mM Tris-Cl, 137 mM NaCl, 25 mM β-glycerophosphate, 2 mM NaPPi, 1 mM Na₃VO₄, 10% glycerol, 2 mM EDTA, 10 μM leupeptin, 0.77 μM aprotinin, 0.5 mM DTT, 10 mM PMSF, and 2 mM benzamidine; pH 7.4) 33 . Cell lysates were centrifuged at 15 000 × g for 10 min and supernatants harvested. The protein concentration of the cell lysate was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Twenty to thirty μg of each lysate was analyzed by Western blot analysis using an antibody specific to active, phosphorylated p38 or JNK. Immunoblots were reprobed with an anti-β-actin antibody as a loading control. The intensity of the active p38 or JNK was quantified using ImageJ software, version 1.37v (National Institutes of Health), and normalized to the intensity of the loading control β-actin.

Data were obtained from three independent experiments of at least triplicate determinations. Statistical analysis was performed using one-way analysis of variance (ANOVA). The error bars represent the standard error of mean (SEM).

To evaluate the effect of A. pleuropneumoniae serotype 10-derived ApxI on proinflammatory cytokine gene expression, porcine alveolar macrophages (PAMs) were incubated with 0-2 CU/mL of ApxI for 2 h and subjected to real-time quantitative PCR (RT-qPCR) analysis. Significant elevation of mRNA levels of IL-1β, IL-8 and TNF-α was noted in PAMs treated with 0.2 to 2 CU/mL of ApxI (p < 0.001) (Figure 1A). The levels of cytokine mRNA increased as the concentration of ApxI elevated to 1 CU/mL, indicating a concentration-dependent effect of ApxI. Substantial levels of mRNA expression were noted in cells treated with ≥ 0.5 CU/mL (Figure 1A). In subsequent experiments, ApxI at 0.5 or 1 CU/mL was used throughout.

In addition, cytokine expression was compared in the absence and presence of an LPS-blocker polymyxin B (PMB). It was noted that 1 CU/mL ApxI preparation without PMB elicited mRNA expression of IL-1β, IL-8, and TNF-α at a level ~8-, 2-, and 13-fold higher than that treated with ApxI in the presence of PMB, respectively (Figure 1A).

To assess the kinetics of ApxI on proinflammatory cytokine gene expression, PAMs were treated with 1 CU/mL of ApxI for varied time periods and mRNA levels were evaluated using RT-qPCR. The mRNA levels of IL-1β, IL-8 and TNF-α elevated at 1 h following ApxI treatment (Figure 1B). The maximal levels of mRNA expression of all three cytokines were observed at 4 h. At 8 or 12 h, the mRNA levels of all these cytokines were less than 63% of the maximal levels (Figure 1B). Collectively, these findings demonstrate A. pleuropneumoniae serotype 10-derived ApxI induces IL-1β, IL-8 and TNF-α gene expression in PAMs in a concentration- and time-dependent manner.

To demonstrate cytokine gene expression in PAMs is truly attributable to the bioactivity of ApxI, ApxI was heat-inactivated (ΔApxI) or pre-incubated with an antiserum raised against a recombinant subunit ApxI protein 27 before application to PAMs and subjected to RT-qPCR analysis. PAMs treated with ΔApxI had mRNA level of IL-1β, IL-8 or TNF-α that was not significantly different from cells without treatment (Figure 2). Moreover, PAMs treated with serum-neutralized ApxI showed a 70-84% attenuation in IL-1β, IL-8 or TNF-α mRNA expression level, indicating IL-1β, IL-8 or TNF-α gene expression is ascribed to the bioactivity of ApxI.

To confirm the effects of ApxI on the protein level of cytokine induction, PAMs were incubated with 1 CU/mL ApxI for 4 h and cell culture supernatants analyzed using quantitative ELISA. Incubation of ApxI resulted in secretion of ~650 pg/mL of IL-1β, 30 ng/mL of IL-8, and 3.2 ng/mL of TNF-α from PAMs (Figure 3). Cells stimulated with ΔApxI had a level of 84 pg/mL of IL-1β in culture supernatant that was not significantly different from cells without treatment (Figure 3A). A similar pattern was also observed for the protein levels of IL-8 and TNF-α in cells treated with ΔApxI and non-treated cells (Figures 3B and 3C). Interestingly, only the basal level (~120 pg/mL) of IL-6 was detected in ApxI-treated PAMs, which was similar to that in cells without treatment or treated with ΔApxI (Figure 3D).

A plethora of studies indicate members of MAPK family play important roles in proinflammatory cytokine induction 21 22 23 24 25 . These findings prompted us to examine the roles of MAPK members p38 and JNK in ApxI effect. PAMs stimulated with 0.5 CU/mL of ApxI for 1 h were subjected to Western blot analysis for active p38 and JNK. ApxI treatment induced an appreciable increase in both active, phosphorylated p38 and JNK in PAMs (Figure 4A). Low levels of phosphorylated p38 were noted in cells treated with ΔApxI that did not significantly differ from non-treated control cells, and there was no detectable phosphorylated JNK in ΔApxI-treated or control cells, suggesting the activation of JNK and p38 MAPK is attributable to ApxI.

To further assess the role of p38 or JNK in ApxI-induced cytokine expression, PAMs were pre-treated with an inhibitor specific to p38 (SB20358) or JNK (SP600125), or vehicle DMSO for 1 h, followed by stimulation with 0.5 CU/mL ApxI for 2 h, and subjected to RT-qPCR analysis. Treatment of p38 inhibitor prior to ApxI stimulation significantly attenuated IL-1β mRNA level by 83%. However, the JNK inhibitor did not attenuate the mRNA expression of IL-1β (Figure 4B). Similarly, ApxI-induced IL-8 mRNA expression was inhibited by p38 inhibitor up to ~85%, but not by JNK inhibitor (Figure 4C). Distinguishably, both p38 and JNK inhibitors blocked ApxI-induced TNF-α mRNA expression by 92% and 54%, respectively (Figure 4D). To confirm our findings, the cytokines secreted from PAMs pre-treated with inhibitors and stimulated with ApxI were also evaluated by quantitative ELISA. In the presence of the p38 inhibitor, the protein levels of IL-1β, IL-8 and TNF-α were significantly inhibited by 69-95% compared to cells without inhibitor pre-treatment and stimulated with ApxI (Figure 4E). Notably, the JNK inhibitor attenuated the protein level of TNF-α, but not IL-1β or IL-8 (Figure 4E). These data suggest that MAPK p38 plays a major role in ApxI-induced IL-1β, TNF-α, and IL-8 expression, while JNK participates only in ApxI-induced TNF-α expression.

The common subunit CD18 of β2 integrins is necessary for A. pleuropneumoniae ApxIII-induced leukolysis 19 . To examine whether ApxI-induced MAPK activation was attributable to ApxI-β2 integrin interaction, a blocking antibody of porcine CD18 was applied to PAMs 1 h prior to stimulation with 0.5 CU/mL ApxI and analyzed by Western blot analysis. In the presence of CD18 antibody, ApxI-induced p38 activation was significantly inhibited by 20-30% (Figure 5A). Cells treated with the control medium or medium containing CD18 antibody displayed only a basal level of phosphorylated p38. Similarly, blocking of CD18 on PAMs resulted in attenuation of ~65% ApxI-activated JNK phosphorylation (Figure 5B). However, phosphorylated JNK was not observed in cells treated with the control medium or medium containing CD18 antibody. Further, to evaluate the effect of ApxI-β2 integrin interaction on cytokine mRNA expression, PAMs were pre-incubated with CD18 antibody, stimulated with 0.5 CU/mL ApxI for 2 h, and subjected to RT-qPCR analysis. Blocking of CD18 molecule attenuated IL-1β, IL-8 and TNF-α mRNA levels by 82-95%. Cells treated with control medium or medium containing CD18 antibody displayed similar basal levels of IL-1β, IL-8 and TNF-α mRNA (Figure 5C). Collectively, these results indicate CD18 plays a pivotal role in ApxI-mediated MAPK activation and expression of proinflammatory cytokine genes in PAMs.