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Exp. Anim. 50(2), 139–145, 2001

In Vitro Migratory Responses of Swine Neutrophils to Actinobacillus pleuropneumoniae Fun-In WANG1), Jay W.J. YANG1), Steven Y. HUNG1), and In-Jen PAN2)

1)Department

of Veterinary Medicine, National Taiwan University, 142 Chou-San Road, Taipei 106, Taiwan and 2)Laboratory of Veterinary Epizootiology, Nihon University 1866 Kameino, Fujisawa 252-8510, Japan

Abstract: Swine neutrophils were quantitatively examined for the direct and indirect migratory responses to Actinobacillus pleuropneumoniae (APP) in vitro and the effects of pseudorabies virus (PrV), frequently co-infecting with APP, were also observed. About 30% of swine neutrophils responded to viable APP, while 3.2% of the neutrophils responded to 0.1% casein which served as the control. The migration to APP was not affected by preincubation of neutrophils with PrV, which inhibited the random migration. When the random migration was normalized to 1, the chemotactic indices for APP, opsonized-APP and casein were 64, 70 and 8.5, respectively. Heat-killed APP or E. coli lipopolysaccharide stimulated the production of interleukin-8 activity by adherent peripheral blood mononuclear cells (PBMC). Preincubation of PBMC with PrV inhibited the production of neutrophil attractant activity when stimulated with heat-killed APP. The results suggested that the direct chemotaxis of neutrophils to viable APP might contribute to early infiltration in Actinobacillus pleuropneumonia, and that PrV might inhibit indirect recruitment of neutrophils to infected lungs by compromising the functions of PBMC. Key words: Actinobacillus pleuropneumoniae, chemotaxis, neutrophil, pseudorabies virus, swine

Introduction Actinobacillus pleuropneumonia is a worldwide distributed disease affecting pigs of all ages. The lesions are characterized by necrosis, hemorrhages and neutrophil infiltration at the early stage and later by vascular thrombosis, edema and macrophage infiltration with fibrinous pleurisy [2, 16, 26]. In the acute phase, swine polymorphonuclear cells (PMN), mostly neutrophils, show chemotaxis to the affected foci and interact with

APP, showing opsonized phagocytosis, oxidative burst, intracelluar bactericidal activity, cytotoxic effects of exotoxins [3, 4, 10, 12, 13, 28], as well as the indirect recruitment of inflammatory cells by cytokines [1, 8, 11]. However, the migratory responses of neutrophils to APP have not been studied in detail [11]. High incidences of APP complication were clinically revealed in swine herds infected with pseudorabies virus (PrV), hog cholera, and porcine reproductive and respiratory syndrome [14, 29]. The bacterial complica-

(Received 28 April 2000 / Accepted 18 September 2000) Address corresponding: F.-I. Wang, Department of Veterinary Medicine, National Taiwan University, 142 Chou-San Road, Taipei 106, Taiwan

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tion in PrV-infected herds has been largely ascribed to the impairment of pulmonary alveolar macrophages [14] and blood mononuclear cells [29], and other herpes viruses are also known to affect neutrophil adhesion and motility. For example, herpes simplex virus (HSV) enhances PMN adhesion to infected cells, in a process which is mediated by cell-derived factors [23, 25]. The PMN chemotaxis is markedly reduced in HSV recurrent patients [22], and HSV infected cells may release leukotriene B4 to enhance chemotaxis [23]. Bovine herpes virus type 1 (BHV-1) induces a significant reduction of neutrophil chemotaxis in vivo [6, 18, 20], but not in vitro [19]. Although a limited susceptibility of swine PMN to PrV has been suggested [21, 29], functional impairment might be induced by PrV as in other herpes viruses. The object of this study was (1) to evaluate in vitro the direct and indirect migratory responses of swine neutrophils to APP, and (2) to observe the effects of PrV on the neutrophil responses to APP. Materials and Methods Animals Heparinized blood (10 U/ml) was obtained from 8to 20-week-old (L × D) pigs born and raised at the National Taiwan University Swine Research Farm. The animals were weaned at 4 weeks of age. Bacteria Colonies of APP serotype 1 grown on chocolate agars were scraped into phosphate-buffered saline (PBS), washed twice, and resuspended in PBS. For calculating colony forming units (CFU), 10-fold serially diluted suspensions were streaked onto agars, which were incubating at 37°C overnight, and counting was made with plates with >10 and 97% were viable. Swine PBMC Heparinized blood was diluted with an equal volume of HBSS, and the dilution was layered onto an equal volume of Histopaque®1.077 g/cc (Sigma) and centrifuged at 600 g for 30 min at 25°C [29]. Mononuclear cells in the interphase were recovered, washed 2 or 3 times with 15 ml of cold HBSS, centrifuged at 650 g for 10 min, and resuspended in RPMI-1640 medium supplement with 2% fetal calf serum (FCS), which yielded >95% viable cells. The PBMC were cultured in 6-well plates at 4–6 × 106 cells/3 ml/well, allowed to adhere at 37°C in 5% CO 2 for 2.5 hr, and then the supernatant and nonadherent cells were removed. The adherent PBMC were washed twice with medium, and cultured in 2 ml of RPMI-1640 supplement with 10% FCS at 37°C for 7 days. This yielded approximately 1 × 106 adherent PBMC of which >90% were viable. For the production of neutrophil attractants, heat-killed APP at a ratio of 50:1 or LPS at 1 µ g/ml were added as stimulants and the culture was made in 2 ml of MEMBSA for 12 hr. The supernatants containing attractant activity were collected, and after centrifugation at 600 g for 10 min to remove cell debris, undiluted or 1/100 diluted samples were tested for chemotaxis. The supernatants were tested for interleukin-8 (IL-8) activity by neutralization with or without mouse anti-pig IL-8 monoclonal antibody (Mab, Endogen) at 8.32 ng/µl at 37°C for 45 min and mixed by tapping occasionally. Microchamber assay The microchamber (Neuro Probe® Inc., Cabin John, MD) filter chemotaxis assay for swine PMNs was a modification of that described by Falk et al. and Harvath et al. [5, 9]. PMNs were suspended in RPMI-1640 medium supplemented with 1% BSA at 2 × 106 cells/ ml. The upper wells contained 50 µl of PMN suspension (100,000 cells/well) and the lower contained 25 µl

of attractants (5 × 107 CFU/well bacteria or indicated concentrations of reagents). Both wells were separated by a polycarbonate membrane with an exposed area of 8 mm2 and 5 µm size pores, which was free of polyvinylpyrrolidone (PVPF-PC, Nucleopore®, Corning, NY). The chambers were incubated at 37°C for 60 to 90 min in humid air with 5% CO2. After incubation, the filter was removed from the chambers, nonmigrated cells remaining on the top surface of the filter were wiped off, and then the filter was fixed in methanol and stained with Riu’s method [15], a Romanowsky-type stain. Neutrophils migrated onto the lower surface of the filter were counted on five selected fields equivalent to 1.124 mm2 with an Olympus BX-50 light microscope at 400x magnification. Tests were made in triplicate and the average number of migrated neutrophils was obtained. The number of neutrophils migrated to RPMI1640 medium was regarded as the random migration number, and the chemotactic index (CI) was calculated by dividing the number of neutrophils migrated to test attractants. The total % of responsive neutrophils was calculated as follows: [(total number of chemotactically migrated neutrophils in five counted fields × 7.117)/ total number of input cells] × 100%. Statistical analysis Results were examined for their significance (p1000:1, the unstained nuclei and rounding and swelling of migrated neutrophils were seen possibly because of the cytotoxic effect of APP. Accordingly, a ratio of 500:1 was used throughout the study. The kinetics of PMN migration were tested with incubation times varying from 15 to 90 min, using viable APP as the attractant. Migration was first apparent at 45 min, increased slowly up to 60 min, then sharply

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Table 1. Chemotactic responses of swine neutrophils to various stimulants Parameter

Random migration

Response to APP

Response to NPS-APPa)

Response to 0.1% Casein

% of responsive cells Chemotactic index

0.8 ± 0.84 (N=21) 1.0 ± 0.00 (N=19)

30.9 ± 11.17 (N=21) 64.0 ± 53.34 (N=19)

28.0 ± 3.10 (N=5) 69.7 ± 38.61 (N=5)

3.2 ± 0.94 (N=4) 8.5 ± 4.33 (N=4)

a) Normal pig serum-opsonized APP.

Table 2. Effect of PrV on swine neutrophil migration to Actinobacillus pleuropneumoniae PrV treatment (MOI)a)

Random migrationb)

Chemotaxis to APPb)

Control (N=6) 0.4 (N=4) 4 (N=6) 40 (N=6)

1752 ± 1039.4 1564 ± 310.8 1836 ± 1328.4 1102 ± 609.6

37323 ± 10230.3 37045 ± 6894.5 38592 ± 12130.3 41317 ± 8855.7

Chemotactic indexc) 24.1 ± 6.47 24.5 ± 7.47 37.2 ± 32.21 49.8 ± 30.14§

PrV effect on Random migrationd)

Chemotaxisd)

1.00 ± 0.00 1.30 ± 0.60 1.01 ± 0.52 0.67 ± 0.31*

1.00 ± 0.00 1.11 ± 0.08 1.03 ± 0.12 1.12 ± 0.10

a) Multiplicity of infection. b) Total numbers of migrated cells in the 8-mm2 are presented. c) Number of migrated cells in each treatment/number of migrated cells in random migration. d) Number of migrated cells in PrV treated sample/ number of migrated cells in control untreated samples. *Indicates a statistically significant difference from that of the control (p=0.025). §Indicates a marginally significant difference from that of the control (p=0.077).

between 60 and 75 min, and peaked at 75 min, thereafter showing a plateau up to 90 min (data not shown), while the cytotoxic effect of APP was evident after incubation for >75 min. Next, viable (non-treated) organisms and NPS-opsonized, heat-killed or formaldehyde-fixed organisms were tested. The viable and NPS-opsonized bacteria attracted on average 30.9 ± 11.17% (N=21) and 28.0 ± 3.10 % (N=5) of PMN, respectively (Table 1). The chemotactic indices were 64.0 ± 53.34 (ranging from 32.1 to 129.3) for viable APP and 69.7 ± 38.61 (ranging from 29.4 to 125.8) for NPS-opsonized APP, respectively. On the other hand, with the heat-killed and formaldehyde-fixed APP the number of migrated neutrophils was much lower than in the random migration. In the next experiment, bacteria and their culture supernatants (N=5 for each) were separately assayed for a reaction time of 35 min. Interestingly, the culture supernatants of heat-killed bacteria attracted a number of neutrophils. Responses of swine neutrophils to FMLP and casein Since there were considerable variations in data

(Tables 1 and 2), two commonly used peptides namely FMLP and casein were tested for attractant effects at concentrations of 10–4 to 10–10 M for FMLP and of 0.001 to 5% for casein. At concentrations of 10–8 or more FMLP elicited no significant migration response of PMN from any individual pig. With casein, a biphasic response at 0.1% and 1% was observed (data not shown), and 3.2 ± 0.94% of neutrophils were responsive, showing an average chemotactic index of 8.5 ± 4.33 (Table 1). The effect of PrV on swine neutrophil migration to APP Various doses of PrV were tested for their effect on the PMN random migration and migration to APP. With increased doses of PrV, the random migration of PMN was decreased, while the chemotactic index was increased, but the migration of PMN to APP remained unaffected (Table 2). As shown in Table 2, there was a significant difference in the random migration between 40 MOI (p=0.025) and without. A marginally significant difference was seen in the chemotactic index to APP between 40 MOI (p=0.077) and without.

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IN VITRO CHEMOTAXIS OF SWINE NEUTROPHILS

Table 3. Production of interleukin-8 activity by adherent PBMC stimulated with heat-killed APP Chemotaxis to IL-8b) (N=3)

Monoclonal antibody treatmenta) Control (Random migration) Anti-IL-8 Mab only Undiluted supernatant only Undiluted supernatant + Anti-IL-8 Mab 1/100 diluted supernatant only 1/100 diluted supernatant + Anti-IL-8 Mab

1243 ± 1158 ± 18038 ± 14520 ± 6024 ± 1364 ±

Chemotactic Indexc) (N=3) 1.0 ± 0.00 0.9 ± 0.26 14.5 ± 1.26 11.6 ± 1.36§ 4.9 ± 0.70 1.1 ± 0.42*

78.4 318.6 2641.7 2619.0 828.7 557.9

a) The amount of anti-IL-8 monoclonal antibody used was 0.208 µg/25 µl/well. b) Total numbers of migrated cells in the 8-mm2 are presented. c) Number of cells migrated to supernatant with or without anti-IL-8 Mab treatment/number of migrated cells in control untreated samples. * Indicates a statistically significant difference from that without Mab treatment (p=0.0014). § Indicates a marginally significant difference from that without Mab treatment (p=0.056).

Table 4. Effect of PrV on the production of neutrophil attractant activity by adherent PBMC stimulated with heat-killed APP PrV treatment (MOI)a)

Positive control (N=4) Mock-4e) (N=3) 4 (N=3) Mock-40e) (N=3) 40 (N=3) 120 (N=3)

Chemotaxis to supernatant of PBMCc)

Chemotaxis to supernatant of PBMC stimulated with APPc)

Chemotactic index in supernatant of PBMCd)

Chemotactic index in supernatant of PBMC stimulated with APPd)

NDb) 1238 ± 648.4 1820 ± 595.1 1497 ± 596.5 2168 ± 1160.9 1810 ± 101.0

6087 ± 3505.0 2923 ± 1146.4 2040 ± 105.6 3649 ± 1814.9 2142 ± 714.4 2375 ± 992.0

NDb) 1.5 ± 0.73 1.7 ± 1.35 1.8 ± 0.71 2.8 ± 1.58 2.3 ± 0.15

7.6 ± 4.85 4.1 ± 0.14 2.0 ± 1.36* 5.3 ± 0.67 2.4 ± 0.37* 3.0 ± 1.15

a) Multiplicity of infection. b) Not done. c) Total numbers of migrated cells in the 8-mm2 are presented. d) Number of migrated cells in each treatment/number of migrated cells in random migration (743 ± 221.9, N=3). e) Supernatants of mock-infected cell culture were added in the same volume as virus suspension required for each dose of PrV treatment. * Indicates a statistically significant difference from that of the mock-infected control (p=0.0002 for 4 MOI and p=0.0028 for 40 MOI).

Production of interleukin-8 activity by adherent PBMC stimulated with heat-killed APP The kinetics of IL-8 production was tested with incubation times varying from 4 to 20 hr post-stimulation, using heat-killed APP as the stimulant. The neutrophil attractant activity was apparent in the cultured supernatant at 4 hr, peaked at 12 hr and thereafter showed a plateau until 20 hr (data not shown). Different stimulants were also tested, in which live APP used at a ratio of 500:1, and killed APP used at ratios of 10:1, 50:1 and 100:1 induced the production of attractant activity equivalent to or higher than that of 1 µg/ml of LPS (data not shown). The IL-8 activity was confirmed by neutralizing the supernatants with mouse anti-pig IL-8 Mab. As shown in Table 3, there was a significant

difference in the chemotactic index to 1/100 diluted supernatant with and without Mab treatment (p=0.0014), and a marginally significant difference in the chemotactic index to undiluted supernatant was also seen with and without Mab treatment (p=0.056). Effect of PrV on the production of neutrophil attractant activity by adherent PBMC stimulated with heat-killed APP As shown in Table 4, there were a significant decreases in the neutrophil attractant activity in supernatant of PBMC preincubated with PrV at 4 and 40 MOI (p=0.0002 and 0.0028, respectively), compared with the respective mock-infected controls.

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Discussion The results suggested that the recruitment of neutrophils directed to viable APP contribute to the initial inflammatory infiltration in the affected swine lung, since about 30% of swine blood neutrophils responded in vitro to viable APP and its metabolites. The attractants evaluated herein seemed rather to be a mixture including bacterial metabolites, which were more efficient than the bacteria themselves. The negative response of PMN to heat-killed and formaldehyde-fixed bacteria indicated that viable APP was required to initiate the sequential events of inflammatory response. The supernatant of heat-killed bacteria suspension attracted neutrophils, in contrast to no effects of bacterial preparations [11], probably due to the degraded bacterial components included [10]. The results of microchamber chemotaxis assay might vary dependent on the doses applied, incubation times, pore sizes of the membrane filter and bacterial strains. Casein served as the best chemo-attractant for swine neutrophils while FMLP didn’t act as attractant in this study. This is consistent with the observations of Fletcher et al. [7] and Thoren-Tolling [27] in which migration distance was measured rather than the number of responsive cells using an agarose assay. The chemotactic index of casein was 8.5 in the present study, which is comparable with 6.31 in the agarose assay [24]. The mechanism of inhibition of neutrophil random migration by PrV is unclear, but the chemotaxis of PMN to APP was not affected by the presence of PrV. The inhibition of random migration was not due to a lowered viability of neutrophils, because the cytotoxic effect of APP was negligible in the present assay, while a limited susceptibility of swine PMN to PrV was indicated [29]. PrV might inhibit neutrophil motility or migration, and the susceptibility of preactivated or primed neutrophils to PrV remains to be determined. The results also suggested that the indirect recruitment of neutrophils via IL-8 also contributes to early inflammatory infiltration in affected swine lungs, since PBMC stimulated with killed APP produced neutrophil attractant activity within 12 hr, a result consistent with the observation of Baarsch et al. [1]. The kinetics and the amount of IL-8 production by adherent PBMC peaking by 12 hr were also comparable with the results of

Lin et al. [17]. PBMC produced IL-8 using killed APP as a stimulant at a ratio of 10:1, while the direct chemotaxis of neutrophils to APP required viable bacteria at a ratio of >100:1. Because 3 to 6 hr were required to produce IL-8 after LPS stimulation [17], IL-8 might play an important role in recruiting neutrophils into the lungs in the first 4–12 hr postinfection, a time when PrV exerts its inhibitory effects on PBMC which is considered to impair innate resistance to APP. The results suggested that direct chemotaxis of neutrophils to viable APP might contribute to the early inflammatory lesions in infected swine. Although the PrV-APP synergism remained unclear, the APP complication in PrV-infected animals is likely related to the impairment of PBMC functions in the indirect recruitment of neutrophils. Acknowledgments This work was supported by a grant 89-ST-6.1-BQ61(31) (NTU grant number 089A-1128) from the Council of Agriculture, Taiwan, R.O.C. References 1. Baarsch, M.J., Scamurra, R.W., Burger, K., Foss, D.L., Maheswaran, S.K., and Murtaugh, M.P. 1995. Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae. Infect. Immun. 63: 3587– 3594. 2. Chen, S.D. 1997. Characterization of polymorphic mononuclear cell in pig Actinobacillus pleuropneumonia. Proc. Natl. Sci. Counc. R.O.C., part B. 21: 1–7. 3. Cruijsen, T.L., Van Leengoed, L.A., Dekker-Nooren, T.C., Schoevers, E.J., and Verheijden, J.H. 1992. Phagocytosis and killing of Actinobacillus pleuropneumoniae by alveolar macrophages and polymorphonuclear leukocytes isolated from pigs. Infect. Immun. 60: 4867–4871. 4. Dom, P., Haesebrouck, F., Kamp, E.M., and Smits M.A. 1992. Influence of Actinobacillus pleuropneumoniae serotype 2 and its cytolysins on pig neutrophil chemiluminescence. Infect. Immun. 60: 4328–4334. 5. Falk, W., Goodwin, R.H., and Leonard, E.J. 1980. A 48well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J. Immunol. Meth. 33: 239–247. 6. Filion, L.G., McGuire, R.L., and Babiuk, L.A. 1983. Nonspecific suppressive effect of bovine herpesvirus type 1 on bovine leukocyte functions. Infect. Immun. 42: 106– 112. 7. Fletcher, M.P., Stahl G.L., and Longhurst, J.C. 1990. In vivo and in vitro assessment of porcine neutrophil activation

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