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Veterinary Immunology and Immunopathology 155 (2013) 155– 161

Contents lists available at SciVerse ScienceDirect

Veterinary Immunology and Immunopathology

j ourna l h omepa ge: www.elsev ier .com/ locate /vet imm

esearch paper

ycoplasma hyopneumoniae-derived lipid-associatedembrane proteins induce apoptosis in porcine alveolaracrophage via increasing nitric oxide production, oxidative

tress, and caspase-3 activation

angfang Baia, Bo Nia, Maojun Liua, Zhixin Fenga, Qiyan Xionga, Shaobo Xiaob,uoqing Shaoa,∗

Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering andechnology, Ministry of Agriculture, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014, ChinaDivision of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhonggricultural University, Wuhan 430070, China

a r t i c l e i n f o

rticle history:eceived 22 January 2013eceived in revised form 17 June 2013ccepted 11 July 2013

eywords:ycoplasma hyopneumoniae

ipid-associated membrane proteinsorcine alveolar macrophagepoptosisitric oxide

a b s t r a c t

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumoniain swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the mainpathogenicity factors in mycoplasma diseases. In this study, we investigated the effectsof M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apop-totic features, such as chromatin condensation and apoptotic bodies, were observed inLAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number ofTUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. Inaddition, flow cytometric analysis using dual staining with annexin-V-FITC and propidiumiodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis inPAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated

aspase-3 caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased productionof nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic ratesincrease and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is sug-gested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophagevia NO production, superoxide anion production, and caspase-3 activation.

Abbreviations: LAMP, lipid-associated membrane proteins; PAM,orcine alveolar macrophage; IL, interleukin; TNF, tumor necrosis fac-or; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nicknd labeling; DAPI, 4′-6-diamidino-2-phenylindole; NO, nitric oxide;OS, reactive oxygen species; NAC, N-acetylcysteine; l-NMMA, N-onomethyl-l-arginine.∗ Corresponding author at: Key Lab of Animal Disease Diagnose of Chi-ese Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsugriculture Academy of Science, 50 Zhongling Street, Nanjing 210014,hina. Tel.: +86 2584390334; fax: +86 2584391973.

E-mail address: guoqing shao@163.com (G. Shao).

165-2427/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.vetimm.2013.07.004

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Mycoplasma hyopneumoniae as the etiological agent ofenzootic pneumonia of swine causes considerable eco-nomic losses in the swine industry worldwide. This tinyorganism binds to the cilia of epithelial cells in theairways and elicits the dysfunction of cilia, leading toimpaired ciliary clearance (DeBey et al., 1992; DeBey

and Ross, 1994). Following infection with M. hyopneumo-niae, swine is susceptible to secondary bacterial or viralinfections, which contribute to significant morbidity andmortality.

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156 F. Bai et al. / Veterinary Immunology

M. hyopneumoniae not only causes physical damage onthe respiratory tract but also modulates the host immuneresponse (Thacker, 2004; Into et al., 2004; Wu et al.,2008). The immunopathological changes associated withmycoplasmal pneumonia are characterized by an infil-tration of the peribronchiolar and perivascular areas bymononuclear leukocytes (Straw, 2006). M. hyopneumo-niae also further initiates immune reactions by inducingmacrophages to produce a number of proinflammatorycytokines, including interleukin (IL)-1�, tumor necrosisfactor (TNF)-�, IL-6, IL-8 and IL-18 both in vivo (Munetaet al., 2008) and in vitro (Damte et al., 2011; Hwang et al.,2008). In addition, M. hyopneumoniae infection has reducedphagocytosis of macrophages upon secondary pathogenexposure in swine (Caruso and Ross, 1990). Accordingly,M. hyopneumoniae infection alters host immune response,resulting in tissue injury and diseases. However, the exactmechanism underlying immunopathogenesis of M. hyop-neumoniae is lacking.

Lipid-associated membrane proteins (LAMP) at the sur-face of mycoplasma not only mediate immune responses(Wise and Kim, 1987) but also induce either necrosis orapoptosis in monocytes and macrophages (Rawadi, 2000).Apoptosis occurs in cells via a sequence of distinct bio-chemical and molecular events. Reactive oxygen species(ROS) have been implicated in apoptotic cascade and trig-ger the activation of initiator and executioner caspases(Beere and Green, 2001; McGowan et al., 1996). In addi-tion, nitric oxide (NO) as a multifunctional molecule isinvolved in a variety of physiological and pathologicalprocesses. NO at low concentrations can protect cellsagainst apoptosis, whereas excessive production of NOtriggers apoptosis in various cell types. Moreover, manymycoplasma species trigger apoptosis by inducing exces-sive NO (Dusanic et al., 2012; Obara and Harasawa, 2010).Altogether, increased ROS, NO production, and the acti-vation of caspases are important molecular events in theapoptotic cascade.

Since little is known about the pathogenic effects of M.hyopneumoniae on porcine macrophages, the present studyaimed to characterize the mechanism underlying the cyto-toxicity of M. hyopneumoniae. Here, we demonstrated thatthe LAMP of M. hyopneumoniae induces ROS and NO pro-duction, and caspase-3 activation, leading to the apoptosisin porcine alveolar macrophage.

2. Materials and methods

2.1. Cell culture

PAM 3D4/21 cell line was purchased from the AmericanType Culture Collection (ATCC CRL-2843) and was main-tained in RPMI 1640 medium (Invitrogen) supplementedwith 10% FBS, 1% antibiotic–antimycotic solutions (Gibco),and 10 mM HEPES (Invitrogen) in a humidified incubatorwith 95% air/5% CO2 at 37 ◦C.

2.2. Mycoplasma culture and the preparation of LAMP

M. hyopneumoniae strain 232 (ATCC 27714) was cul-tivated in KM2 medium until the beginning of the

unopathology 155 (2013) 155– 161

stationary phase. Then it was collected after centrifu-gation at 12,000 × g for 10 min and the preparation ofLAMP was performed as described previously (Rawadiand Roman-Roman, 1996). Briefly, LAMPs of pathogenicM. hyopneumoniae in Triton X-114 phase fraction wereprecipitated by methanol and then were resuspended insterile PBS. The protein concentration was determinedby using a BCA protein assay kit (Beyotime Biotech.,Jiangsu, China). The protein extracts were incubated with100 �g/ml polymyxin B for 2 h to eliminate the possibilityof endotoxin contamination from protein solution. LAMPswere collected and stored at −80 ◦C until further use.

2.3. MTT-based viability test

The MTT assay is an in vitro colorimetric assay for mea-suring cell viability and proliferation. This assay is based onthe conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into purple formazan by themitochondrial enzymes in viable cells, and the intensity ofpurple is proportional to the number of viable cells. PAM3D4/21 cells (1 × 104 cells/well; volume: 90 �l) in 96-wellplates were allowed to grow for 4 h. After incubation ofthe cells with 0–0.6 mg/ml LAMP for 48 h, the cytotoxicitywas determined by MTT cell viability/cytotoxicity assay kit(Beyotime Biotechnology, Jiangsu, China). Briefly, 10 �l theMTT solution was added to each well and the cells wereincubated for 4 h at 37 ◦C. The formazan precipitates (pur-ple crystals) formed after incubation was solubilized byadding 100 �l dissolved solution of formazan. After solubi-lization of the formazan precipitates, the absorbance wasmeasured at a wavelength of 570 nm using a microplatereader (BioTeck, EL-340, USA).

2.4. DAPI staining

PAM 3D4/21 cells (1 × 105 cells/well) in 24-well plateswere allowed to grow at 37 ◦C for 12 h. Subsequently, thecells were exposed to LAMP (0.4 mg/ml) for 24 h, followedby fixation in 4% paraformaldehyde (4 ◦C, 25 min) and per-meabilization with 0.1% Triton X-100 (25 ◦C, 10 min). Afterwashing twice with PBS (pH 7.4), the cells were stainedwith DAPI (0.5 �g/ml, Roche, Germany) and observedunder a fluorescence microscope (Nikon, Eclipase E600,Tokyo, Japan).

2.5. TUNEL assay

PAM 3D4/21 cells (1 × 105 cells/well) were seeded in 6-well plates and exposed to LAMP (0.4 mg/ml) for 12, 24 and36 h. A TUNEL assay kit (fluorescein in situ cell death detec-tion kit; Roche, Mannheim, Germany) was used to detectapoptotic cells according to the manufacturer’s instruc-tion. As the positive control, the fixed and premeabilizedPAM 3D4/21 cells were incubated with DNase I recombi-

10 min at 25 ◦C to induce DNA strand breaks, prior to TUNELstaining. The negative control was incubated in label solu-tion without transferase. And the cells were observed usinga fluorescence microscopy (Nikon, Eclipase E600).

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apoptotic bodies by DAPI staining (Fig. 2). In addition,DNA fragmentation, which is a significant hallmark ofapoptosis, was monitored by the TUNEL assay in LAMP-treated PAM 3D4/21 cells. When the TUNEL assay was

Fig. 1. Effects of different concentrations of LAMP of M. hyopneumoniae onthe survival rates of PAM 3D4/21 cells. PAM 3D4/21 cells were incubated

F. Bai et al. / Veterinary Immunology

.6. NO assay

The NO production was measured using a NO assay kitBeyotime Biotech., Jiangsu, China). After incubation withAMP (0.4 mg/ml) for 12, 24 and 36 h, supernatants of PAMD4/21 cells were mixed with Griess reagent I and Griesseagent II, and then the absorbance was read at 550 nmbackground absorbance at 630 nm) using a microplateeader (BioTeck, EL-340, USA).

.7. Measurement of superoxide anion generation

Superoxide anion levels were measured using auperoxide assay kit (Beyotime Biotech.) following man-facturer’s instruction. Briefly, the cells (1 × 105 cells/well)rom exponentially growing cultures were seeded in 96-ell culture plate. After washing with PBS, the cells were

ncubated with 200 �l superoxide assay solution includ-ng WST-1 and catalase at 37 ◦C for 3 min. Then the cells

ere incubated with indicated concentration of LAMP.fter incubation for 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and2 h, absorbance was read at a wavelength of 450 nm with

microplate reader (BioTeck, EL-340, USA).

.8. Annexin-V-FITC-PI staining

The exposure of phospholipid phosphatidylserine (PS)o the external leaflet of the plasma membrane in earlypoptosis can be detected by annexin-V-FITC and deadells can be detected by PI. Annexin-V-FITC-PI stainingllows differential analysis of live, apoptotic, and dead cellsBossy-Wetzel and Green, 2000). PAM 3D4/21 cells (1 × 105

ells/well) were grown in culture medium with or without-NMMA (0.5 mM) or NAC (10 mM) for 1 h. Then the cellsere treated with LAMP (0.4 mg/ml) or M. hyopneumoniae

106 color change unit (CCU)/ml] for 12, 24 and 36 h. Thenhe cells were harvested by trypsinization and were pel-eted by centrifugation. After washing with a binding buffer10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4),he cells were stained with annexin-V-FITC (final concen-ration of 0.25 �g/ml, room temperature, 10 min) and wereubsequently stained with propidium iodide (PI, final con-entration of 0.5 �g/ml, 4 ◦C, 5 min) in the dark. Data wereollected using a flow cytometry system (Becton and Dick-nson, USA) and analyzed using FlowJo software (TreeStar,nc., Ashland, USA).

.9. Caspase-3 assay

The caspase-3 activity was determined using a caspase- activity assay kit (Beyotime Biotech.) following manu-acturer’s instruction. Briefly, PAM 3D4/21 cells (1 × 105

ells/well) were grown in culture medium with or with-ut l-NMMA (0.5 mM) or NAC (10 mM) for 1 h. Then theells were grown in culture medium with or without LAMP0.4 mg/ml) for 12 h. Cells were collected and lysed usingysate buffer. The cell lysate was centrifuged (12,000 × g,

◦C for 15 min), and then 40 �l of the supernatant (cyto-

olic extracts, 100 �g) was mixed with 50 �l of reactionuffer and 10 �l of Ac-DEVD-pNA (a caspase-3 colorimetricubstrate). After incubation at 37 ◦C for 2 h, the absorbance

unopathology 155 (2013) 155– 161 157

was measured with a plate reader at a wavelength of405 nm (BioTeck, EL-340, USA).

2.10. Statistical analysis

Data represent the mean ± standard deviation (SD) ofat least three individual experiments. Statistical differencewas determined by the Student’s t-test or one-way ANOVAfollowed by Turkey test. P values < 0.05 were consideredstatistically significant.

3. Results

3.1. Effects of M. hyopneumoniae LAMP on the survivaland proliferation of PAM 3D4/21 cells

The effects of the LAMP on the survival and prolif-eration of PAM 3D4/21 cells were analyzed by the MTTassay. Cell survival rates of PAM 3D4/21 cells incubatedwith 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/ml LAMP for 48 hwere 97.54 ± 3.7%, 92.71 ± 4.4%, 75.8 ± 14%, 50.7 ± 22%,28.89 ± 15% and 13.96 ± 0.81%, respectively (Fig. 1). Addi-tionally, M. hyopneumoniae LAMP inhibited the survivalof PAM 3D4/21 cells with an IC50 value of 0.4 mg/ml andthe concentration was used in the subsequent assays. Theresults indicated a dose-dependent cytotoxicity of M. hyop-neumoniae LAMP on PAM 3D4/21 cells.

3.2. M. hyopneumoniae LAMP induced apoptotic bodiesand TUNEL positive cells in PAM 3D4/21 cells

Apoptosis is characterized by various typical mor-phological changes. To confirm the apoptotic effect, themorphological changes of M. hyopneumoniae LAMP-treated PAM 3D4/21 cells were evaluated using the DAPIstaining. After exposing to LAMP for 12 h, 24 h and 36 h,PAM 3D4/21 cells exhibited chromatin condensation and

with series concentrations of LAMP and the survival rates were analyzedby the MTT assay. All values were normalized to control, in which PAM3D4/21 cells were incubated in RPMI 1640 medium without LAMP. Theone-way ANOVA followed by Turkey test was used to compare the survivalrates of treatment- groups and control-group. *P < 0.05, **P < 0.01.

158 F. Bai et al. / Veterinary Immunology and Immunopathology 155 (2013) 155– 161

Fig. 2. Apoptotic features in LAMP-treated PAM 3D4/21 cells weredetected using DAPI and TUNEL staining. As a positive control, the fixedand permeabilized PAM 3D4/21 cells were treated with DNase I recombi-nant before the TUNEL staining. The negative control of TUNEL assay wasperformed without transferase. After incubation with 0.4 mg/ml LAMPfor 12, 24 and 36 h, typical apoptotic bodies in PAM 3D4/21 cells weredetected by the DAPI staining, and the TUNEL positive cells were observedin PAM 3D4/21 cells. Besides, the cells in the same microscopic view werevisualized using bright-field light microscope. Representative microscopyimages are shown (scale bars indicate 50 �m). The rectangle delineated by

Fig. 3. Effects of LAMP of M. hyopneumoniae on NO production and super-oxide anion production in PAM 3D4/21 cells. (A) Concentration of NOwas determined by the Griess assay in PAM 3D4/21 cells incubated withLAMP of M. hyopneumoniae for 6, 12, 18 and 24 h. (B) Levels of superoxideanion were quantified in PAM 3D4/21 cells exposed to LAMP for 15 min,

the white solid line shows a high magnification view of the area indicatedby the arrowhead.

performed, negative control and positive control wereincluded. Negative control was incubated in label solutionwithout transferase; as a positive control, PAM 3D4/21cells treated with DNase I before the TUNEL assay. Theresults indicated LAMP significantly increased the num-ber of TUNEL positive cells at 12 h, 24 h and 36 h whencompared with untreated control (Fig. 2).

3.3. M. hyopneumoniae LAMP induced the production ofNO in PAM 3D4/21 cells

NO is a crucial signal molecule and plays a very impor-tant role during apoptosis. The production of NO wasquantified by a colorimetric assay measuring the accu-mulation of the NO end products, nitrite and nitrate, inthe medium. Compared with untreated control, NO con-centrations in the supernatants of PAM 3D4/21 cells afterincubation with M. hyopneumoniae LAMP for 6 h, 12 h,18 h and 24 h were 1.37-, 1.57-, 1.81-, 2.42-fold increases,respectively (Fig. 3A). Thus, NO level in PAM 3D4/21 cellsdisplayed a time-dependent increase following incubationwith M. hyopneumoniae LAMP.

3.4. M. hyopneumoniae LAMP increased the superoxideanion levels in PAM 3D4/21 cells

ROS can function as signaling molecules and play acritical role in mediating apoptosis. To determine the

effects of M. hyopneumoniae LAMP on ROS production,we measured the superoxide anion (a primary source ofROS) production in PAM 3D4/21 cells after incubationwith LAMP. Compared with untreated control, incubation

30 min, 1 h, 2 h, 4 h, 8 h and 12 h. Data are presented as mean ± SD of threereplicates. *P < 0.05 and **P < 0.01 versus control.

with LAMP for 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 hinduced 2.53-, 2.98-, 3.58-, 2.51-, 2.47-, 2.51- and 3.07-fold increases, respectively, in the superoxide anion levelsin PAM 3D4/21 cells (Fig. 3B). The results demonstratedthat LAMP significantly increased ROS production in PAM3D4/21 cells.

3.5. M. hyopneumoniae LAMP induced apoptosis in PAM3D4/21 cells in a time-dependent manner

Loss of plasma membrane asymmetry is one of theearliest features of apoptosis. Membrane phosphatidylse-

rine (PS) is originally located in the inner leaflet of theplasma membrane. During apoptosis, the membrane PS istranslocated to the outer leaflet and exposed to the extra-cellular environment. Annexin V can specifically bind to

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he exposed PS with a high affinity, and therefore candentify apoptosis at early stage when it is conjugated

ith FITC. Moreover, the membranes of dead and dam-ged cells, other than intact membranes of viable cells,llow the penetration of PI. The percentages of apop-

ig. 4. The apoptosis of PAM 3D4/21 induced by LAMP was assessed byow cytometry using annexin-V-FITC-PI staining. (A) PAM 3D4/21 cellsere stimulated with 0.4 mg/ml of LAMP for 12 h, 24 h and 36 h. After

equentially staining with annexin-V-FITC and PI, the cells were analyzedy flow cytometry and representative dot plots were displayed. Dot plotsith double negative staining represent living cells (Q4), dot plots withositive staining for annexin-V-FITC and negative staining for PI repre-ent the early apoptotic cells (Q3), and dot plots with double-positivetaining represents the late apoptotic cells (Q2). (B) Quantification of apo-tosis of PAM 3D4/21 cells were incubated with LAMP (0.4 mg/ml) or M.yopneumoniae [106 color change unit (CCU)/ml] for 12, 24 and 36 h afterreatment with or without l-NMMA (0.5 mM) or NAC (10 mM) for 1 h. Theesults are mean ± SD from three independent experiments. * P < 0.05 and*P < 0.01 versus control; #P < 0.05 and ##P < 0.01 versus LAMP only-treatedells.

unopathology 155 (2013) 155– 161 159

totic cells were examined by flow cytometry after stainingwith annexin-V-FITC and PI. The results obtained in threeindependent experiments showed that the percentagesof apoptotic cells (annexin-V+PI+ and annexin-V+PI−) ofthe three time-points were 42.3 ± 7.9%, 52.9 ± 8.2% and82.7 ± 11.3%, respectively (Fig. 4). In agreement with theeffect of M. hyopneumoniae LAMP, live M. hyopneumoniaeitself also induced apoptosis in a time-dependent mannerin PAM 3D4/21 cells (Fig. 4B).

Moreover, to determine whether the apoptosis inducedby LAMP is dependent on the NOS activation/superoxideanion production, PAM 3D4/21 cells were treated withantioxidant NAC or iNOS inhibitor l-NMMA prior to LAMPtreatment. LAMP-induced increase in the apoptotic per-centages was inhibited by prior treatment with NAC orl-NMMA in all these time-points (Fig. 4B). The resultsrevealed that LAMP induced a time-dependent productionof superoxide anion and NO, resulting in apoptosis in PAM3D4/21 cells.

3.6. Activation of caspase-3 was induced by LAMP inPAM 3D4/21 cells

Caspase-3 is a key mediator of apoptosis. To inves-tigate the possible involvement of downstream effectorcaspases in the apoptosis process triggered by LAMP, theactivity of caspase-3 was measured in LAMP-treated PAM3D4/21 cells. Exposure of PAM 3D4/21 cells to LAMP for12 h resulted in a 2.36-fold increase of caspase-3 activitywhen compared with untreated controls (Fig. 5). Moreover,treatment with 10 mM NAC or 0.5 m l-NMMA decreasedthe LAMP-induced caspase-3 activation (Fig. 5). The results

indicated that LAMP triggers caspase-3 activation throughenhanced superoxide anion production and NO synthaseactivation.

Fig. 5. Effects of LAMP of M. hyopneumoniae on caspase-3 activation inPAM 3D4/21 cells. Caspase-3 activity was quantified in PAM 3D4/21 cellstreated with LAMP for 12 h prior to treatment with or without l-NMMA(0.5 mM) or NAC (10 mM) for 1 h. Data are presented as mean ± SD of threereplicates. *P < 0.05 and **P < 0.01 versus control; #P < 0.05 versus LAMPonly-treated cells.

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4. Discussion

We firstly reported that LAMP of M. hyopneumoniaeinduced apoptosis in PAM 3D4/21 cell line, which wasdependent on the increased NO production, ROS levels, andcaspase-3 activation.

Base on MTT results, the concentration of LAMP(0.4 mg/ml) that caused 50% inhibition (IC50 value) of cellsurvival was used in the experiments. The range of con-centrations of LAMP (0.1–0.6 mg/ml) used in this studycorresponds to approximately 2.5 × 106–1.5 × 107 CCU/mlof M. hyopneumoniae. These concentrations were physi-ologically relevant, based on previous studies in which5 × 106–5 × 107 CCU/ml of M. hyopneumoniae was existingin bronchoalveolar lavage fluids during in vivo infection(Shibata et al., 1998). We studied the effect of live M.hyopneumoniae on the PAM 3D4/21 cells, and the live M.hyopneumoniae also induced the cellular apoptosis in atime-dependent manner. Moreover, we have tested andfound that LAMP (0.4 mg/ml) also induced apoptotic bod-ies formation in the primary porcine alveolar macrophagesisolated from bronchoalveolar lavage fluids (data notshown), suggesting the continuous PAM 3D4/21 cell lineis a suitable cell model system to study the interactionbetween the M. hyopneumoniae and macrophages.

Two mechanisms of eukaryotic cell death, necrosisand apoptosis, have thus far been identified. Cells under-going necrosis are morphologically characterized by theswelling of organelles, disrupted plasma membrane andcellular explosion. By contrast, apoptosis (programmed celldeath) is characterized by several major stereotypic mor-phological features such as cell shrinkage, condensationof the nuclear chromatin, and apoptotic-body formation.The cytopathogenicity of several mycoplasma species hasalready been studied (Dusanic et al., 2012; Obara andHarasawa, 2010; Wu et al., 2008). Moreover, membranelipoproteins of Mycoplasma genitalium, Mycoplasma fer-mentans and Mycoplasma salivarium induce apoptosis inseveral lymphocytic and monocytic cell lines (Into et al.,2002; Wu et al., 2008). In this study, apoptotic body for-mation was observed in LAMP-treated PAM 3D4/21 cells.Furthermore, exposure to LAMP induced a time-dependentapoptosis in PAM 3D4/21 cells. These findings stronglyindicated that LAMP kills PAM 3D4/21 cells by inducingapoptosis rather than necrosis.

NO generated from l-arginine is an important messen-ger participating in various physiological and pathophysi-ological processes (Korhonen et al., 2005). NO can eitherpromote or inhibit apoptosis in various cells (Kim et al.,1999). Apoptosis in human B lymphocytes is inhibited bylow dose of NO generated by nitric oxide synthase (NOS)(Mannick et al., 1994). By contrast, excessive NO reactswith superoxide anion radicals to form peroxynitrite, lead-ing to the activation of apoptosis cascade (Korhonen et al.,2005). Previous studies have shown that M. hyopneu-moniae, Mycoplasma hyorhinis and Mycoplasma synoviaestimulate excessive production of NO production in RAW

264.7 cell line, human gastric carcinoma AZ-521 cell line,and chicken chondrocytes, respectively (Dusanic et al.,2012; Hwang et al., 2008; Obara and Harasawa, 2010). Con-sistently, we demonstrated that LAMP of M. hyopneumoniae

unopathology 155 (2013) 155– 161

significantly increased NO production in PAM 3D4/21 cellsover time during apoptosis. In addition, treatment withNOS inhibitor inhibited LAMP-induced apoptosis of PAM3D4/21 cells. The results indicated that LAMP of M. hyop-neumoniae induced apoptosis in PAM 3D4/21 cells throughNO production.

During apoptosis, mitochondria are the main site ofgeneration of ROS. Oxidative stress occurs when excessiveproduction of ROS overwhelms the capability of cellularantioxidant defense system (Curtin et al., 2002). In pre-vious study, LAMP from Mycoplasma pneumoniae inducesan increase in the ROS levels in pulmonary epithelial A549cells (Choi et al., 2012). This present study showed a sub-stantial increase in the superoxide anion levels in PAM3D4/21 cells exposed to LAMP of M. hyopneumoniae andthe superoxide anion inhibitor inhibited LAMP-inducedapoptosis, indicating that LAMP-induced apoptosis in PAM3D4/21 cells is dependent on intrinsic mitochondria-dependent pathway.

Excessive ROS lead to mitochondrial dysfunction andthen trigger the activation of caspase cascades and apopto-sis (Lu et al., 2011). Caspase, a family of cysteine proteases,plays a major role in the intracellular signal pathway inapoptosis. Caspases generally consist of the upstream ini-tiator caspases such as caspase-2, -8, -9 and -10, andthe downstream effector caspases such as caspase-3, -6and -7 (Shi, 2002). In many animal cells, the execu-tion phase of apoptosis correlates with the activation ofcaspase-3 (Kuida et al., 1996; Miura et al., 1993). Moreover,M. hyorhinis activates caspase-3 in human gastric carci-noma AZ-521 cells (Obara and Harasawa, 2010). In thisstudy, treatment of PAM 3D4/21 cells with LAMP inducedthe caspase-3 activation, which was inhibited by eithersuperoxide anion inhibitor or NOS inhibitor. These resultsindicated that LAMP-induced caspase-3 activation path-way is dependent on NO and ROS production in PAM3D4/21 cells.

LAMP promotes the development of mycoplasma-associated diseases by interfering with the host immunesystem (Razin et al., 1998). However, the molecular basisof mycoplasma pathogenicity remains unclear. Here wedemonstrated that LAMP of M. hyopneumoniae inducedapoptosis in the PAM 3D4/21 cell line. Excessive apopto-sis in immune cells results in immunosuppression and theincreased susceptibility to reinfection, which associatedwith increased severity of diseases (Thacker et al., 1999).Moreover, the apoptosis induced by pathogens further ini-tiates inflammation (Torchinsky et al., 2010). Accordingly,LAMP of M. hyopneumoniae might induce macrophagesapoptosis, impairing the swine immune system. As aconsequence, M. hyopneumoniae successfully colonizelung tissue, contributing to the respiratory diseases inswine.

In conclusion, our results clearly demonstrated thatLAMP derived from M. hyopneumoniae induced apoptosisin porcine alveolar macrophage cell line through enhancingthe production of NO, superoxide burst, and activation of

caspase-3. These findings provide insights concerning theeffects of mycoplasma infection on immune reactivity andpave the way for novel therapeutic strategies to preventtissue damage in mycoplasma-associated diseases.

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onflict of interest statement

None of the authors of this paper has a financial or per-onal relationship with other people or organizations thatould inappropriately influence or bias the content of theaper.

cknowledgements

This work was supported by grants from the Nationalatural Science Foundation of China (No. 31100135), arant from Jiangsu Agriculture Science and Technologynnovation Fund (CX(11)4038).

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