Vol. 60, No. 11

INFECMION AND IMMUNI, Nov. 1992, p. 4867-4871

0019-9567/92V114867-05$02.00/0 Copyright © 1992, American Society for Microbiology

Phagocytosis and Killing of Actinobacillus pleuropneumoniae by Alveolar Macrophages and Polymorphonuclear Leukocytes Isolated from Pigs TOINE L. M. CRUIJSEN,l.2* LEO A. M. G. VAN LEENGOED,1'2 TOOS C. E. M. DEKKER-NOOREN,2 ERIC J. SCHOEVERS,1 'AND JOS H. M. VERHEIJDEN' Department ofHerd Health and Reproduction, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, 1 and Department of Herd Health, Pathology and Epidemiology, Central Vetennary Institute, Lelystad,2 The Netherlands Received 10 March 1992/Accepted 21 August 1992

To study the cellular response of phagocytic cells to Actinobacillus pleuropneumoniae, we investigated whether porcine alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) are able to phagocytize and intracellularly kill A. pleuropneumoniae in vitro. Bacterial cultivation methods of A. pleuropneumoniae were used to assess in vitro phagocytosis and the ability to kill. A specific-pathogen-free pig was killed, blood was collected, and PMN were isolated and counted. The AM were isolated by lung lavage of the same animal and counted. In addition, convalescent-stage serum was collected from a specific-pathogen-free pig that was infected with A. pleuropneumoniae. Both porcine AM and porcine PMN effectively phagocytized A. peuropneumoniae in the presence of convalescent-stage pig serum. PMN killed 90 to 99%o of the bacteria intracellularly, whereas AM did not. Because A. pkuropneumoniae produces exotoxins that kill porcine AM and porcine PMN, we incubated equal amounts of bacteria and phagocytic cells and tested the viability of the cells 120 min later. In the presence of convalescent-stage pig serum, A. pleuropneumoniae was toxic to AM but not to PMN. Probably in porcine AM, intracellular released toxins of A. pleuropneumoniae lessen the ability of the cell to kill the bacterium. Consecutive lysis of AM and release of viableA. pleuropneumoniae may initiate the characteristic porcine pleuropneumonia.

Actinobacilluspleuropneumoniae causes severe and often fatal hemorrhagic necrotizing pleuropneumonia in pigs. Pulmonary lesions are characterized by granulocytes, lymphocytes, macrophages, hemorrhages, necrosis, exudation of fibrin, and fibrin deposits in blood vessels and lymphatics (2, 6, 15, 21, 22, 24). To understand the pathogenesis of this disease, knowledge of both the etiological agent and the host defense mechanism against inhaled lung pathogens is required. Alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) form the major defense mechanism of the distal airways against invading bacteria (25). Sibille and Reynolds (25) suggested that when in vivo AM are unable to clear an inoculum of bacteria, a PMN influx in the alveoli is required for clearance. In porcine models, small doses (80 CFU) of A. pleuropneumoniae inoculated deep in the bronchi induced pleuropneumonia (28). This inflammatory response with influx of exudate and PMN suggests that porcine AM are unable to clear the inoculum. So, knowledge of the abilities of AM and PMN to phagocytize and kill A. pleuropneumoniae will allow a better understanding of factors that modulate the inflammatory response of the porcine lung to A. pleuropneumoniae. In this study, we compared the in vitro phagocytosis and killing abilities of AM and PMN isolated from the same specific-pathogen-free (SPF) pig. BecauseA. pleuropneuwoniae produces cytolysins that affect AM as well as PMN (1, 20, 27) and because opsonins are required for efficient phagocytosis (26), we used convalescent-stage pig serum in

MATERIALS AND METHODS Isolation and preparation of porcine PMN. PMN were isolated from heparinized blood from 10- to 14-week-old clinically healthy SPF pigs (Central Veterinary Institute breeding colony). Six experiments were conducted, and in each experiment, a pig was anesthetized with 300 to 500 mg of Na-pentobarbital and exsanguinated. A 300-ml amount of heparinized (10 U/ml) blood was collected. Two volumes of pig blood were mixed with 1 volume of 6% (wt/vol) dextran (70,000 molecular weight) in physiologic saline (0.9% NaCl in distilled water), and the mixture was incubated for 60 min at 37C. The leukocyte-rich supernatant fluid was harvested and centrifuged for 10 min at 180 x g. The resulting pellet was suspended in phosphate-buffered saline (PBS; 0.123 M NaCl, 0.01 M Na2HPO4, 0.0032 M KH2PO4; pH 7.2), and 4 ml of the suspension was layered onto 3 ml of FicollHypaque suspension (Ficoll-Paque; Pharmacia, Uppsala, Sweden) and centrifuged for 30 min at 400 x g. The resulting pellet was washed with NH4C1 solution (8.26 g of NH4C1, 1.09 g of NaHCO3, 0.037 g of Na2EDTA in 1,000 ml of distilled water) and centrifuged for 5 min at 120 x g. The supematant fluid was discarded, and the remaining erythrocytes in the pellet were lysed with a cold (4°C) NH4C1 solution for 5 min. The suspension was centrifuged for 5 min at 120 x g, and the supernatant fluid was discarded. The pellet was washed twice with PBS and suspended in Eagle minimal essential medium (EMEM) supplemented with Earle salts, L-glutamine, and 6% SPF-pig serum (EMEMSPF). Cells were counted in a hemocytometer and adjusted to 107 cells per ml of EMEM-SPF. Viability of cells was determined by nigrosine dye exclusion (7), and cells were used only when the percentage of viable cells exceeded 95%. Isolation and preparation of porcine AM. Porcine AM were

our test system.

*

Corresponding author. 4867

4868

CRUIJSEN ET AL.

obtained from the lungs of the same SPF pigs that were used for blood collection. Lung lavage samples were collected as described by van Leengoed et al. (27). Cells were suspended in EMEM-SPF and adjusted to 107 cells per ml. Viability of the cells was determined, and cells were used only when the number of viable cells exceeded 95%. Preparation of opsonins. Convalescent-stage pig serum was collected from a 14-week-old SPF pig that was endobronchially inoculated (28) at 8 weeks of age with 5 x 102 CFU of A. pleuropneumoniae 13261, the reference strain of serotype 9 (16). Six weeks after inoculation, blood samples were collected. Blood was allowed to clot, and serum was then collected, stored in small aliquots, and frozen at -70°C. Serum was tested in a 13-mercaptoethanol tube agglutination test (12), which indicates the presence of opsonins in the serum. We also tested the convalescent-stage serum in hemolysin and cytotoxin neutralization assays as described by Kamp and van Leengoed (8). Neutralization titers were expressed as the reciprocal of the lowest dilution showing less than 50% hemolysis or 50% stained macrophages. SPF-pig serum. A 10-week-old SPF pig was exsanguinated, and serum was collected and stored in small aliquots at -70°C. The serum was thawed shortly before use. This serum was also tested in the ,3-mercaptoethanol agglutination test and the hemolysin and cytotoxin neutralization tests. Preparation of bacterial suspension. A. pleuropneumoniae 13261 (16) was cultured for 6 to 8 h on sheep blood agar with NAD (SBV) plates. Bacterial growth on each plate was rinsed off with 5 ml of EMEM, and the number of CFU was determined by plating 10-fold dilutions on SBV plates. The bacterial suspension was stored overnight at 4°C. When suspensions are stored at 4°C, the number of CFU per milliliter and the 50% lethal dose for mice prove to be constant for at least 48 h (27). Next morning, the colonies were counted, and the suspension was diluted with EMEM to a concentration of approximately 107 CFU/ml. Opsonization of A. pleuropneumoniae. A. pleuropneumoniae was opsonized by adding 120 pl of convalescent-stage serum to 2 ml of A. pleuropneumoniae suspension (2 x 107 CFU) in a siliconized glass tube. Glass tubes were closed with nontoxic silicon rubber stoppers and incubated for 30 min at 37°C in a head-over-head rotor at 6 rpm (test tube rotator; Breda Scientific, Breda, The Netherlands). Phagocytosis assays. Phagocytosis assays were conducted as described by Leijh et al. (10). Briefly, 2 x 107 AM or PMN in EMEM-SPF and 2 x 107 opsonizedA. pleuropneumoniae were combined in a siliconized glass tube. The tube was placed in a 37°C incubator and rotated at 6 rpm. At 0, 15, 30, 60, and 90 min, a 0.5-ml sample was collected and mixed with 1.5 ml of ice-cold EMEM to stop phagocytosis. Phagocytes were pelleted for 4 min at 110 x g and 4°C. Serial 10-fold dilutions of the supernatant fluid were made, and each dilution was vortexed (5 s; 2,500 rpm) to break up clumped bacteria. The spiral-gradient agar dilution method was used to determine the number of CFU by plating 100-,ul samples of the dilutions on SBV plates (Spiral System Instruments, Bethesda, Md.). To evaluate the bactericidal effect of serum, opsonized A. pleuropneumoniae samples without AM or PMN were used as controls. The degree of phagocytosis was expressed as the percent decrease in the initial number of viable extracellular A. pleuropneumoniae. This value was corrected for the bactericidal effect of the serum that occurs during the same period (10). Killing assay. Because the degree of bacterial killing had to be determined independently of the degree of ingestion,

INFEcr. IMMUN.

phagocytes were allowed to ingest a maximum number of A. pleuropneumoniae before being used in the killing assay. The time needed for phagocytes to ingest a maximum number ofA. pleuropneumoniae was determined as follows. Equal volumes of a phagocyte suspension and the opsonized bacterial suspension were mixed and incubated under rotation (6 rpm) at 37°C. Every minute, samples were collected and immediately mixed with ice-cold EMEM. Phagocytes were pelleted, washed twice (4 min, 110 x g, 4°C), and suspended in 2 ml of PBS that contained 0.1% Triton X-100 at 37°C. To enhance lysis, phagocytic cells were vortexed for 5 s at 2,500 rpm and incubated for 10 min at room temperature (18°C). Then, tubes were vortexed for 15 s at 2,500 rpm, and the number of ingested bacteria was determined by plating 10-fold dilutions on SBV plates. The killing assay was described in detail by Leijh et al. (10). A 2-ml sample of a phagocyte suspension and 2 ml of opsonized A. pleuropneumoniae were mixed and incubated at 37°C under continuous rotation at 6 rpm. The PMN were incubated for 1 min, and the AM were incubated for 10 min. Ice-cold EMEM was added to stop phagocytes from ingesting and killing the bacteria. Phagocytes were then washed twice (4 min, 110 x g, 4°C) to remove extracellular A. pleuropneumoniae. After the second wash, the pellet was suspended in 4 ml of EMEM with 6% convalescent-stage pig serum at 37°C. Tubes were incubated at 37°C with rotation at 6 rpm. After 0, 15, 30, 60, and 90 min, 0.5-ml samples were collected and mixed with 1.5 ml of ice-cold EMEM to stop further intracellular killing. These samples were centrifuged for 4 min at 110 x g at 4°C, and the pellet of phagocytic cells was lysed as described above. The number of CFU in the suspension was determined. To ascertain that the macrophages used were competent to kill ingested bacteria, we compared both Staphylococcus aureus (type 42 D) (10)loaded macrophages and A. pleuropneumoniae-loaded macrophages in one killing assay. V'iability of phagocytic cells. Viability of AM and PMN was determined by nigrosine dye exclusion (7) at 120 min after the first mixing of phagocytes and A. pleuropneumoniae in the phagocytosis assay. In the killing assay, viability of the cells was determined at 120 min after the killing assay was started. Statistical analysis. Standard variance analysis was performed with Genstat 5 (18) using a log count of A. pleuropneumoniae as the dependent variable with time effect, cell-type effect (AM or PMN), and interaction effect (time x cell type) as independent variables. RESULTS Characterization of convalescent-stage pig serum and SPFpig serum. The (-mercaptoethanol agglutination titer of the convalescent-stage pig serum was 60. The hemolysin neutralization titer of the convalescent-stage serum was 512, and the cytotoxin neutralization titer was 2,048. Titers of SPFpig serum were undetectable in the 3-mercaptoethanol agglutination test and the hemolysin and cytotoxin neutralization assays. Phagocytosis. The number of A. pleuropneumoniae in control samples (bacteria and serum without phagocytic cells) decreased slightly during incubation (Fig. 1 and 2). In the presence of AM, the number of A. pleuropneumoniae decreased 10-fold 15 min after phagocytes and A. pleuropneumoniae were mixed (Fig. 1). Thereafter, the number of A. pleuropneumoniae did not decrease. In the presence of PMN, the number of A. pleuropneumoniae decreased five-

PHAGOCYTOSIS AND KILLING OF A. PLEUROPNEUMONIE

VOL. 60, 1992

1071

4869

- N.,

- .-L..

E a

_ control

6 10 -

c

AM~

~

~

~

I

E

C

O

-E

5

10

1

CIO

-

0 Uc

IL. 105_-

0 103

104

PMN

C

0 3 10 0

10

20

30

40

50

60

70

80

90

100

time (nin)

0

FIG. 1. Kinetics of phagocytosis of A. pleuropneumoniae by porcine AM. Equal volumes (2 ml) of 107 AM per ml in EMEM-SPF and 107A. pleuropneumoniae per ml suspended in EMEM with 6% convalescent-stage pig serum were combined and incubated at 37°C at 6 rpm (head-over-head rotor). Control experiments were run simultaneously by combining 2 ml of 107 bacteria per ml (in EMEM with 6% convalescent-stage pig serum) and 2 ml of EMEM-SPF. Samples (0.5 ml) were taken at 0, 15, 30, 60, and 90 min and added to 1.5 ml of ice-cold EMEM. Resulting samples were centrifuged (4 min, 110 x g, 4°C), and the number of CFU per milliliter in the supernatant fluids was determined by plate counting. Bars are standard deviations (n = 6).

fold during incubation (Fig. 2). AM ingested the most bacteria 30 min after the phagocytes were mixed with A. pleuropneumoniae. The average degree of phagocytosis was 92% (range, 86 to 99%) of the initial extracellularA. pleuropneumoniae. At 90 min, the number of ingested A. pleuropneumoniae decreased slightly to 72% (range, 14 to 99%) of the initial extracellular A. pleuropneumoniae. In contrast, the number of A. pleuropneumoniae ingested by PMN increased steadily during the incubation period, and finally, 7 10 -1

10

-

-

-

---i

_S 2PUN

0

IL O

10

-

-

0

10

20

30

40

50

60

70

80

90

100

time (min)

FIG. 2. Kinetics of phagocytosis of A. pleuropneumoniae by porcine PMN. Equal volumes (2 ml) of 107 PMN per ml in EMEMSPF and 107A. pleuropneunoniae per ml suspended in EMEM with 6% convalescent-stage pig serum were combined and incubated at 37°C at 6 rpm (head-over-head rotor). Control experiments were run simultaneously by combining 2 ml of 107 bacteria per ml (in EMEM with 6% convalescent-stage pig serum) and 2 ml of EMEM-SPF. Samples (0.5 ml) were taken at 0, 15, 30, 60, and 90 min and added to 1.5 ml of ice-cold EMEM. Resulting samples were centrifuged (4 min, 110 x g, 4°C), and the number of CFU per milliliter in the supernatant fluids was determined by plate counting. Bars are standard deviations (n = 6).

5

10

15

20

25

0I

30

35

time (min)

FIG. 3. Determination of the optimal ingestion time of AM or PMN for A. pleuropneumoniae as used in the intracellular killing test. Equal volumes (2 ml) of 107 AM or PMN per ml in EMEM-SPF were combined with 2 ml of 107 A. pleuropneumoniae per ml suspended in EMEM with 6% convalescent-stage pig serum and incubated at 37°C at 6 rpm (head-over-head rotor). Samples (0.5 ml) were taken every minute for 10 min and thereafter every 5 min until 30 min and added to 1.5 ml of ice-cold EMEM. Resulting samples were centrifuged and washed twice (4 min, 110 x g, 4°C), and pellets were recovered and lysed in 2 ml of 0.1% Triton X-100 in PBS. The number of CFU per milliliter in the lysed pellet was determined by plate counting.

at 90 min, 93% (range, 70 to 99.9%) of A. pleuropneumoniae had been ingested. Intracellular killing. Figure 3 shows the optimal period of ingestion, during which the maximum number of A. pleuropneumoniae were recovered. The maximum number of viable intracellular A. pleuropneumoniae were recovered after 1 min of incubation in the PMN assay. No decrease of intracellular A. pleuropneumoniae during the first 30 min was seen in the AM assay, and after 10 min, the maximum number was reached. Therefore, intracellular killing assays with PMN and AM were performed 1 and 10 min after phagocytosis of opsonized A. pleuropneumoniae. The PMN killed 91% (range, 80 to 96%) of A. pleuropneumoniae within 15 min of incubation (Fig. 4). After 90 min of incubation, 95% (range, 90 to 99%) of the A. pleuropneumoniae were nonviable. In contrast, AM did not appear to kill A. pleuropneumoniae intracellularly (Fig. 4). AM killed 24% of ingested S. aureus after 15 min. After 120 min, 82% of S. aureus were killed, whereas no A. pleuropneumoniae were killed. The interaction term (time x cell type) in the standard analysis of variance contributed significantly (P < 0.001) to the log count of A. pleuropneumoniae; i.e., the A. pleuropneumoniae count in PMN decreased significantly more than that in AM. V'iability of phagocytes. In our phagocytosis assay, A. pleuropneumoniae was more toxic to AM than to PMN. More than 50% of the AM were dead after 120 min, whereas less than 37% of the PMN were dead. In the killing assay, this toxic effect was more pronounced (Table 1). S. aureus was not toxic to AM in the killing assay.

DISCUSSION We investigated whether porcine AM and PMN are able to phagocytize and killA. pleuropneumoniae in vitro. Both cell types were isolated from the same pig in order to eliminate variation between pigs. Both AM and PMN phagocytizedA.

4870

CRUIJSEN ET AL.

10

7

IN'FECT. IMMUN.

-

.~6

-10

L_

L-

_

IT AM

T

--

-

a

10

1

-

T

I

o 10 10

0

10

20

30

40

50

60

70

80

90

100

time (min)

FIG. 4. Kinetics of intracellular killing of A. pleuropneumoniae by porcine AM or porcine PMN. AM were allowed to ingest A. pleuropneumoniae for 10 min, and PMN ingested bacteria for 1 mi, both at a bacterium/cell ratio of 1:1. Ice-cold EMEM was added to stop phagocytes from ingesting and killing the bacteria. Phagocytes were then centrifuged and washed twice (4 min, 110 x g, 4°C). Thereafter, 2 x 107 macrophages loaded with A. pleuropneumoniae or 2 x 107 PMN loaded with A. pleuropneumoniae were suspended in 4 ml of EMEM with 6% convalescent-stage pig serum and incubated at 370C at 6 rpm (head-over-head rotor). Samples (0.5 ml) were taken (at 0, 15, 30, 60, and 90 min) and added to 1.5 ml of ice-cold EMEM. Resulting samples were centrifuged (4 min, 110 x g, 4"C), and pellets were recovered and lysed in 2 ml of 0.1% Triton X-100 in PBS. The number of CFU per milliliter in the lysed pellet was determined by plate counting. Bars are standard deviations (n = 6).

pleuropneumoniae in the presence of convalescent-stage pig serum. Control samples including nonimmune serum (SPFpig serum) instead of convalescent-stage pig serum were omitted, as we found no ingested bacteria in our killing assay. To check whether A. pleuropneumoniae multiplied during our assay, we included control samples with bacteria and serum but no phagocytes. Our assay medium lacked NAD, which is essential for growth ofA. pleuropneumoniae. During our assay, plate counts revealed no growth of A. pleuropneumoniae. However, in the phagocytosis assay with AM, bacterial counts compared with control counts increased from 30 min onward. With PMN, this relative increase was not seen. This suggests that the observed dead and consequently lysed AM resulted in release of A. pleuropneumoniae ingested earlier but still viable. Therefore, we counted the CFU in the supernatant and pellet until 240 min after the start of the killing assay. From 210 min on, CFU counts of the supernatant increased while those of the pellet decreased. However, counts decreased more rapidly in the TABLE 1. Percentage of dead cells as determined by nigrosine dye exclusion (n = 6) % Dead celis in:

Type of cell

Phagocytosis

assay"

Killing assay' Mean ± SD

Range AM 72 8 63 8 50-73 57-81 PMN 12 11 1-37 8 4 4-15 aCells were tested at 120 min after incubation with opsonized A. pleuroin EMEM with 3% convalescent-stage pig serum (37C). pneumoniae b Mean + SD

Range

Celis loaded with A. pleuropneumoniae were tested at

120 min after

suspension in EMEM with 6% convalescent-stage pig serum (37C).

pellet than they increased in the supernatant. Loss of pelleted AM paralleled inconsistent CFU counts. Our evidence that viable bacteria were released from AM is based on (i) a relative increase inA. pleuropneumoniae counts from 30 min on in our phagocytosis assay, (ii) the inability of AM to kill A. pleuropneumoniae, and (iii) the observed death of AM. In contrast to AM, PMN killed 95% of the ingested A. pleuropneumoniae. The optimal time for ingestion of opsonized A. pleuropneumoniae in the killing assay was 1 min for PMN and 10 min for AM. These different times were used to obtain maximum recovery of viable intracellular bacteria. The maximal number of viable bacteria in PMN was lower than that in AM and is best explained by rapid intracellular killing in PMN. It is unlikely that the defective killing of AM is caused by the higher number of ingested A. pleuropneumoniae, as we could not detect any killing by AM in studies designed to determine the preincubation time. Because the AM used were able to kill S. aureus, we were sure that AM were generally competent at killing. van Leengoed et al. (27) concluded that AM are less susceptible to the cytolytic effect of A. pleuropneumoniae in the presence of serum from convalescent pigs, since convalescent-stage pig serum neutralizes the cytolysins produced by A. pleuropneumoniae (8). Sera of immune pigs completely protect pigs against A. pleuropneumoniae challenge (3). In this study, A. pleuropneumoniae 13261 was cytotoxic for AM even in the presence of convalescent-stage pig serum. That extracellular cytolysin was not completely neutralized in our assay is unlikely, because we used 3% (1:33) convalescent-stage pig serum in our phagocytosis assay with a hemolysin neutralization titer of 1:512 and a cytotoxin neutralization titer of 1:2,048. To ascertain that all extracellular toxins were neutralized, filtered supematant of opsonized A. pleuropneumoniae was combined with S. aureus-loaded macrophages and did not affect killing of S. aureus (unpublished results). Therefore, a more likely explanation is that ingested A. pleuropneumoniae was still toxic for AM, while antibodies in our assay were not able to neutralize cytolysin produced in the cells. This hypothesis fits with the deficient bactericidal activity of AM in our killing assay. Our observation that AM are unable to intracellularly kill A. pleuropneumoniae agrees with the defective intracellular killing of several other bacterial species by murine AM (5, 13). There are at least two possible explanations for the defective killing of A. pleuropneumoniae by AM. First, an impaired oxygen metabolism of AM (19) may be the cause. Second, cytolysin produced by intracellular A. pleuropneumoniae affects cellular killing mechanisms of AM. Inhibition of bacterial killing is also seen after incubation of bovine AM or PMN with large numbers of Pasteurella haemolytica, which also produces an exotoxin (4, 11). In our study, however, killing by PMN was not impaired. PMN generate twice as much superoxide and release five times as much hydrogen peroxide as macrophages do (19). Both oxygen products are highly reactive agents with potential bactericidal capacity. Cytolysin produced by A. pleuropneumoniae might affect AM and cause impairment of oxygen product generation and thus killing of ingestedA. pleuropneumoniae. The antibacterial functions of AM and PMN were determined with a reliable microbiologic method that allows separate assessment of phagocytosis and intracellular killing of bacteria by phagocytes (10). Bacterial counts made in our killing assay give direct information about viability of bacteria, whereas other methods, with measurement of 02 consumption, H202 production, or nitroblue tetrazolium

PHAGOCYTOSIS AND KILLING OF A. PLEUROPNEUMONIAE

VOL. 60, 1992

reduction, are suggested to reflect activity of the killing mechanisms of phagocytes (10). Bacterial resistance to these mechanisms can cause erroneous results. The pathogenesis of pleuropneumonia in pigs is still not well understood. The natural infection route of A. pleuropneumoniae is believed to be aerogenous (14, 17). We suggest that A. pleuropneumoniae bacteria that escape ciliary action and penetrate alveolar spaces are phagocytized by AM. Bacterium-loaded macrophages can enter lung tissue by migrating through the alveolar epithelium or by entering the lymphofollicular aggregates (9). On the basis of our in vitro findings, we postulate that A. pleuropneumoniae will lyse AM instead of being killed. Cell death of AM may actually amplify an inflammatory response, and influx of PMN will follow. PMN finally kill the bacterium. That PMN kill A. pleuropneumoniae is supported by the finding of van Leengoed et al. (28) that A. pleuropneumoniae does not usually pass the bronchial lymph node. This suggests an active role for PMN in preventing bacterial sepsis. Mild, even inapparent disease as well as severe disease that results in rapid death occurs under field conditions (23). The difference between mild and severe cases of pleuropneumonia can be caused by differences in the numbers of bacteria that actually reach the lungs or by variations in neutralizing antibodies against A. pleuropneumoniae cytolysins, but it may also be caused by variations in opsonizing antibodies among pigs. Our observation that A. pleuropneumoniae can survive within AM suggests that the presence of opsonins may enhance the transport of bacteria into lung tissue. The absence of cytolysin-neutralizing antibodies combined with the presence of opsonins may indicate those pigs that are most susceptible to A. pleuropneumoniae. Therefore, serological screening of acute cases of pleuropneumonia under field conditions is indicated.

ACKNOWVLEDGMENTS We thank Hilda Ottema for her technical assistance, Gonnie van Osta for the statistical analysis, and Victoria Thatcher for correcting the manuscript. The helpful discussions of Elbarte Kamp and Uri Vecht are gratefully acknowledged. REFERENCES 1. Bendixen, P. H., P. E. Shewen, S. Rosendal, and B. N. WVikie. 1981. Toxicity of Haemophilus pleuropneumoniae for porcine lung macrophages, peripheral blood monocytes, and testicular cells. Infect. Immun. 33:673-676. 2. Bertram, T. A. 1985. Quantitative morphology of peracute pulmonary lesions in swine induced by Haemophilus pleuropneunoniae. Vet. Pathol. 22:598-609. 3. Bosse, J. T., R. P. Johnson, M. Nemec, and S. Rosendal. 1992. Protective local and systemic antibody responses of swine exposed to an aerosol of Actinobacillus pleuropneumoniae serotype 1. Infect. Immun. 60:479-484. 4. Czuprynski, C. J., H. L. Hamilton, and E. J. Noel. 1987. Ingestion and killing of Pasteurella haemolytica Al by bovine neutrophils in vitro. Vet. Microbiol. 14:61-74. 5. Degre, M. 1969. Phagocytic and bactericidal activities of peritoneal and alveolar macrophages from mice. J. Med. Microbiol. 2:353-357. 6. Hini, H., K. Konig, J. Nicolet, and E. Scholl. 1973. Zur Haemophilus Pleuropneumonie beim Schwein. V. Pathomorphologie. Schweiz. Arch. Tierheilk. 115:191-203. 7. Hudson, L., and F. C. Hay. 1980. Practical immunology, 2nd ed., p. 29-31. Blackwell Scientific Publications Ltd., Oxford. 8. Kamp, E. M., and L. A. M. G. van Leengoed. 1989. Serotyperelated differences in production and type of heat-labile hemolysin and heat-labile cytotoxin of Actinobacillus (Haemophilus) pleuropneumoniae. J. Clin. Microbiol. 27:1187-1191.

4871

9. Lauwerins, J. M., and J. H. Baert. 1977. Alveolar clearance and the role of pulmonary lymphatics. Am. Rev. Respir. Dis. 115:625-683. 10. Leih, P. C. J., R. van Furth, and T. L. van Zwet. 1986. In vitro determination of phagocytosis and intracellular killing of polymorphonuclear and mononuclear phagocytes, p. 46.1-46.21. In D. M. Weir, L. A. Herzenberg, C. Blackwell, and L. A. Herzenberg (ed.), Handbook of experimental immunology, vol 2. Cellular immunology. Blackwell Scientific Publications Ltd., Oxford. 11. Markham, R. J. F., and B. N. Wilkie. 1980. Interaction between Pasteurella haemolytica and bovine alveolar macrophages: cytotoxic effect on macrophages and impaired phagocytosis. Am. J. Vet. Res. 41:18-22. 12. Mittal, K. R., R. Higgins, S. Lariviere, and D. Leblanc. 1984. A 2-mercaptoethanol tube agglutination test for diagnosis of Haemophiluspleuropneumoniae infection in pigs. Am. J. Vet. Res. 45:715-719. 13. Nibbering, P. H., T. van den Barselaar, J. S. van de Gevel, P. C. Leih, and R. van Furth. 1989. Deficient intracellular killing of bacteria by murine alveolar macrophages. Am. J. Respir. Cell. Mol. Biol. 1:417-422. 14. Nicolet, J., H. Konig, and E. Scholl. 1969. Zur HaemophilusPleuropneumonie beim Schwein. Schweiz. Arch. Tierheilk. 111:166-174. 15. Nielsen, R. 1970. Haemophilus parahaemolyticus as the cause of pleuropneumonia in swine. I. Clinical, pathological, and epidemiological studies. Nord. Vet. Med. 22:240-245. 16. Nielsen, R. 1985. Serological characterization of Haemophilus pleuropneumoniae (4ctinobacillus pleuropneumoniae) strains and proposal of a new serotype: serotype 9. Acta Vet. Scand. 26:501-512. 17. Nielsen, R., and M. Mandrup. 1977. Pleuropneumonia in swine caused by Haemophilus parahaemolyticus. A study of the epidemiology of infection. Nord. Vet. Med. 29:465-473. 18. Payne, R. W., P. W. Lane, A. E. Ainsley, K. E. Bicknell, P. G. N. Digby, S. A. Harding, P. K. Leech, H. R. Simpson, A. D. Todd, P. J. Verrier, R. P. White, J. C. Gower, G. Tunnicliffe Wilson, and L. J. Paterson. 1987. Genstat 5 reference manual, p. 389-401. Clarendon Press, Oxford. 19. Reiss, M. R., and D. Roos. 1978. Differences in oxygen metabolism of phagocytosing monocytes and neutrophils. J. Clin. Invest. 61:480-488. 20. Rosendal, S., J. Devenish, J. I. MackInnes, J. H. Lumsden, S. Watson, and H. Xun. 1988. Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae. Am. J. Vet. Res. 49:1053-1058. 21. Sanford, S. E., and G. K. S. Josephson. 1981. Porcine Haemophiluspleuropneumoniae epizootic in southwestern Ontario: clinical, microbiological, pathological and some epidemiological findings. Can. J. Comp. Med. 45:2-7. 22. Schiefer, B., and J. Greenfield. 1974. Porcine Haemophilus parahemolyticus pneumonia in Saskatchewan. II. Bacteriological and experimental studies. Can. J. Comp. Med. 8:105-110. 23. Sebunya, T. N. K., and J. R. Saunders. 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182:1331-1337. 24. Shope, R. E. 1964. Porcine contagious pleuropneumonia. I. Experimental transmission, etiology, and pathology. J. Exp. Med. 119:357-368. 25. Sibille, Y., and H. Y. Reynolds. 1990. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am. Rev. Respir. Dis. 141:471-501. 26. Thwaits, R. N., and S. Kadis. 1991. Immunogenicity of Actinobacillus pleuropneumoniae outer membrane proteins and enhancement of phagocytosis by antibodies to the proteins. Infect. Immun. 59:544-549. 27. van Leengoed, L. A., E. M. Kamp, and J. M. A. Pol. 1989. Toxicity of Haemophilus pleuropneumoniae to porcine lung macrophages. Vet. Microbiol. 19:337-349. 28. van Leengoed, L. A. M. G., and E. M. Kamp. 1989. Endobronchial inoculation of various doses of Haemophilus (Actinobacillus) pleuropneumoniae in pigs. Am. J. Vet. Res. 50:2054-2059.

Phagocytosis and killing of Actinobacillus pleuropneumoniae by alveolar macrophages and polymorphonuclear leukocytes isolated from pigs.

To study the cellular response of phagocytic cells to Actinobacillus pleuropneumoniae, we investigated whether porcine alveolar macrophages (AM) and p...
1MB Sizes 0 Downloads 0 Views