Effect of Pasteurella multocida Toxin on Bone Resorption In Vitro

INFECTION AND IMMUNITY, Dec. 1992, p. 4984-4988 0019-9567/92/124984-05$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 60, No. 12

Effect of Pasteurella multocida Toxin on Bone Resorption In Vitro R.

FELIX,`* H. FLEISCH,1 AND P. L. FRANDSEN2

Department of Pathophysiology, University of Berne, Murtenstrasse 35, CH-3010 Bern, Switzerland, 1 and Biotechnology Centre for Livestock and Fish, National Veterinary Laboratory, DK-1790 Copenhagen V, Denmark Received 7 July 1992/Accepted 25 September 1992

Pasteurella multocida toxin (PMT), which is the primary etiologic factor in the pathogenesis of progressive atrophic rhinitis in pigs, was found to stimulate bone resorption in vitro. This stimulation was observed both in cultures of murine calvaria by measuring the release of calcium and of the lysosomal enzyme 13-glucuronidase and in murine long bone cultures by measuring the release of calcium. Both systems showed the same dose response curve, with the maximal effect at a concentration of 5 ng/ml. The effect on calvaria was studied in more detail. PMT increased bone resorption 24 h after its addition and always had to be present to express an effect. Calcitonin was able to inhibit this increase of resorption completely, and inhibitors of prostaglandin synthesis suppressed it partially. Although the data show an effect of PMT on bone tissue, the results do not exclude an action on cells in the nasal cavity, which could indirectly stimulate bone resorption.

chromatography with an immobilized anti-PMT murine monoclonal antibody by the method of Foged (9). PMT was quantified by enzyme-linked immunosorbent assay (9), and its cytopathic activity was determined in embryonic bovine lung (EBL) cells (29). PMT was free of lipopolysaccharide, as determined by the Limulus test (Struers, Copenhagen, Denmark) (detection limit, 0.01 endotoxin unit per ml). Modified BGJb medium (2) was obtained from Amimed (Basel, Switzerland). Antibiotics were from Seromed (Berlin, Germany). Bovine serum albumin (radioimmunoassay grade) and the prostaglandin inhibitors indomethacin and flurbiprofen were from Sigma Chemical Co. (St. Louis, Mo.). Hydrocortisone was from Fluka (Buchs, Switzerland). 1,25-Dihydroxycholecalciferol [1,25(OH)2D3] was a gift of Hoffmann-La Roche (Basel, Switzerland). Animals. Outbred NMRI mice were used. Bone cultures. (i) Calvaria. Culture conditions used earlier (18, 31) were modified. Instead of culturing the bones on a grid, the bones were incubated in shaking culture dishes. Calvarium explants, consisting of frontal and parietal bones from 5- to 6-day-old mice were obtained aseptically. The bones were divided into two halves along the median suture and preincubated for 1 day in 6-well plates in 2 ml of BGJb medium supplemented with 50 U of penicillin per ml, 50 ,g of streptomycin per ml, and 150 p.g of ascorbic acid per ml. The bones were then cultured in pairs; one half of the bone was used as a control, and the other half was treated. The dishes were incubated on a rocking platform (angle, 350; frequency, 16/min) in 5% C02-95% air at 37°C. For determination of the blank value, four dishes without bones were also incubated. The medium from the dishes was collected after 3 days. In order to prevent precipitation of calcium phosphate during storage, 50 ,ul of 2 M acetic acid was added to the medium. Each bone was put in 4 ml of 5% trichloroacetic acid to dissolve the minerals. Bone resorption was calculated as the amount of calcium released into the medium as a percentage of total calcium (bone and medium) after subtraction of the blank value. The effect of the test substance is given as the difference between the treated bone and the control bone (T - C).

Progressive atrophic rhinitis is a disease of pigs characterized by atrophy of the nasal bones, leading, in the most severe form, to a twisted or shortened snout (4, 6, 11, 30). Pasteurella multocida toxin (PMT), also known as dermonecrotic toxin (10, 22), has been established as the primary etiologic factor in the pathogenesis of progressive atrophic rhinitis in pigs (10, 24). PMT has been purified and consists of a single polypeptide of 146 kDa (4, 9, 22). The PMT gene has been cloned (14, 16, 26), sequenced, and shown to encode 1,285 amino acid residues (3, 17, 25). Data base searches did not show similarities to other known sequences. Based on histological examination of nasal bones after treatment with PMT, various suggestions concerning the mechanism of action have been made. While inhibition of osteogenesis and accelerated osteoclastic bone resorption have been reported (5, 6, 8), others did not observe changes in osteoblasts, only an increase in the number of osteoclasts (21, 22). P. multocida cell extract or culture supernatant added in vitro to murine fetal long bones increased calcium release and the numbers of preosteoclasts and osteoclasts (15). Since these preparations were crude, the actions of other compounds could not be excluded. As it is now possible to purify PMT by affinity chromatography without contamination by lipopolysaccharide, we have studied the action of this polypeptide on bone resorption in calvarium and long bone cultures. Both systems were used because certain factors stimulate bone resorption by different mechanisms in these two kinds of bone (20); e.g., transforming growth factor-, even induces the opposite effects, namely, inhibition and stimulation of bone resorption (27).

MATERIALS AND METHODS PMT was purified from an extract of the toxigenic type D strain P. multocida subsp. multocida NCTC 12178 by affinity *

Corresponding author. 4984

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FIG. 1. Effect of PMT on bone resorption of murine calvaria. After preincubation for 1 day, pairs of calvarium halves were cultured for 3 days in the presence of increasing amounts of PMT or 1 ng of 1,25(OH)2D3 per ml. The release of calcium is given as a percentage of total calcium (bone and medium) and expressed as the difference between treated and control bones (T - C). Each bar shows the difference between five pairs of calvarium halves, given as mean + standard error of the mean (SEM). Values significantly different from that of the control (P < 0.05 [*] and P < 0.001 [***]) are indicated.

Ca was analyzed spectrophotometrically (28) by taking advantage of the fact that Ca bound to methylthymol blue gives a complex with a maximum A612. (ii) Fetal long bones. Pregnant NMRI mice were injected with 50 ,uCi of 45Ca on day 16 of gestation. One day later, the animals were killed. The radii and ulnae of the fetuses were aseptically removed and transferred to Gey's solution (35). The bone explants were precultured in an atmosphere of 5% C02-95% air at 37°C in 12-well plates for 24 h to allow exchange of 45Ca with the medium. The preculture incubation was done in 0.5 ml of BGJb medium containing 10% heat-inactivated fetal calf serum as well as 100 U of penicillin per ml, 100 ,g of streptomycin per ml, and 50 p,g of freshly added ascorbic acid per ml. Thereafter, the bones were transferred to fresh medium containing either control solution or test substance. The medium was changed every 3 days, except for the last 4-day period. After 10 days, the bones were decalcified in 1 ml of 5% trichloroacetic acid. Net 45Ca release was calculated as the difference between Ca release of live bone and of dead bone killed by three cycles of freeze-thawing and expressed as a percentage of the total 45Ca (bones and medium). Glucuronidase. The enzyme activity of glucuronidase was determined as described by Barrett (1). To the assay system 0.2 ml of medium was added to give a final volume of 0.6 ml containing 5 mM 4-nitrophenyl-p-glucopyranosiduronic acid in 0.1 M sodium acetate/acetic acid, pH 5.0. After 23 h of incubation at 37°C, the reaction was stopped by adding 0.6 ml of buffer containing 0.5 M NaHCO3 and 0.5 M Na2CO3. Enzyme activity was proportional to incubation time and amount of medium added. Statistical analysis. Results were analyzed using Student's t test.

RESULTS PMT stimulated bone resorption in calvaria in a dosedependent manner, reaching an optimal effect at approximately 5 ng/ml (Fig. 1). The amount of Ca released was only slightly smaller than that found when the bones were incubated with 1 ng of 1,25(OH)2D3 per ml. A similar dose

0.3 1.25 5

20 80

PMT

1 ng/ml 1,25(OH)2D3

FIG. 2. Effect of PMT on the release of 13-glucuronidase by murine calvaria. For details of the procedure used, see the legend to Fig. 1. Instead of calcium, the release of ,B-glucuronidase into the medium was measured. Enzyme activity released per calvarium halves is expressed as difference (T C) between five pairs of calvarium halves (mean + standard error of the mean [SEM]). Values significantly different from that of the control (P < 0.05 [*] and P < 0.001 [***]) are indicated. -

response curve, with saturation at 5 ng/ml, was observed when the release of a lysosomal enzyme, 13-glucuronidase, from bone to medium was investigated (Fig. 2). The effect of PMT on bone resorption increased with time of exposure. The actual release of Ca and ,-glucuronidase started after 24 h of incubation (Fig. 3). Incubation with PMT for 24 h did not induce bone resorption during the next 48 h and the removal of the toxin after 48 h reduced resorption in the following 24 h (data not shown). This result suggests that PMT has to be present in the culture in order to stimulate bone resorption. The effect of PMT on the release of calcium was blocked by calcitonin and the effect of ,B-glucuronidase was nearly blocked (Table 1). Inhibitors of prostaglandin synthesis inhibited, although not completely, the release of calcium. However, the release of 0-glucuronidase activity was not inhibited or inhibited only to a limited extent (Table 2). PMT also stimulated bone resorption in radii and ulnae (Fig. 4).

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FIG. 3. Time course of the effect of PMT on bone resorption of murine calvaria. After preincubation for 24 h, pairs of murine calvarium halves were cultured for different times in the presence or absence of PMT. Calcium release is expressed as the difference between treated and control halves (T C) and is given as mean standard error of the mean (SEM) for the different time periods. Values significantly different from that of the control (not PMT treated) (P < 0.05 [* or +], P < 0.005 [**], and P < 0.001 [*** or +++]) are indicated. -

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TABLE 1. Effect of calcitonin on PMT-stimulated calcium and Treatment

No calcitonin Calcitonin

0-glucuronidase release in murine calvariaa Release of ,B-glucuronidase (mU/half calvarium)

Release of calcium (%) Without PMT With PMT

2.6 ± 0.5 1.1 ± 0.4*

9.9 ± 2.1 1.6 ± 0.7*

Without PMT

With PMT

0.097 ± 0.013 0.087 ± 0.008

0.245 ± 0.028 0.126 ± 0.006*

a After preincubation for 24 h, bones were cultured for 48 h in the presence or absence of PMT. Five pairs of calvarium halves were either treated with 0.1 mU of calcitonin per ml or not treated with calcitonin. Release of calcium and ,B-glucuronidase is given as mean ± standard error of the mean for each treatment group. Values significantly different from the value obtained for the group not treated with calcitonin (P < 0.05 [*]) are indicated.

The dose-response curve was similar to the one obtained for the calvarium culture (Fig. 1). DISCUSSION

The present results show that affinity-purified PMT is a novel and potent inducer of bone resorption in cultured explants from murine calvaria and long bones. Its maximal activity is comparable to that of 1,25(OH)2 vitamin D3. Both long bones and calvaria had the same dose-response curve to PMT, with the maximal effect at 5 ng/ml. These two types of bone are known to respond differently to certain bone resorption-inducing agents. Tumor necrosis factor alpha and epidermal growth factor stimulate bone resorption in calvaria by increasing prostaglandin synthesis but act by a different mechanism in long bones (20, 33). In the case of transforming growth factor-13, the two systems even respond in opposite ways. This factor stimulates bone resorption in calvaria (32, 34) but inhibits it in long bones (27). The data of the current study show that PMT acts in both systems with similar potencies. Information about the nature of the PMT-induced bone resorption mechanism was obtained in the calvarium cultures. Since the release of lysosomal enzymes is closely linked to bone resorption (7), the appearance of 3-glucuronidase in the medium was also measured. Calcitonin, a hormone which binds to and inhibits osteoclasts (12, 23), blocked bone resorption, thus indicating that the PMTinduced bone resorption is osteoclast mediated. Inhibitors of prostaglandin synthesis at concentrations fully inhibiting the synthesis of prostaglandin (19) decreased the bone resorption without completely inhibiting the process. These data suggest that PMT induces bone resorption in calvarium

cultures by at least two different pathways, namely, the release of prostaglandins and other substances still unknown. Dose-response and time course experiment results for the release of Ca and 3-glucuronidase were similar. These two parameters were, however, inhibited by calcitonin and inhibitors of prostaglandin synthesis in different ways.

This effect of PMT is in agreement with an earlier finding (15) in which conditioned medium from nasal mucosa exposed to extracts of toxigenic P. multocida induced bone resorption in long bones. However, large amounts of conditioned medium seemed to inhibit resorption. In the experiment presented here, no inhibition of bone resorption was found up to a concentration of 100 ng/ml, suggesting that the inhibition was due to substances other than PMT. Both infection with toxigenic P. multocida and injection of purified PMT in pigs leads to atrophy of nasal turbinate bones (4, 10, 13, 30). However, no lesions have been observed in other bone structures after natural infection (30) or after parenteral administration of the purified toxin (4, 10, 13). These observations indicate that the nasal turbinate bones are a specific target of PMT. This might be explained by the fact that bone cells of this tissue, in contrast to other bone tissues, are especially sensitive to PMT. In our study, PMT induced bone resorption to similar degrees in calvaria, which are membranous bones, and in long bones, which are endochondral bones. However, since no nasal bone was tested, a special sensitivity of nasal bone tissue to PMT cannot be excluded. Alternatively, cells other than bone cells present in the nasal cavity might be the target of PMT and be induced to release local factors stimulating bone resorption in this tissue.

TABLE 2. Effect of prostaglandin synthesis inhibitors on PMT-stimulated calcium and ,B-glucuronidase release in murine calvariaa Prostaglandin synthesis inhibitor

Calcium release (%) Calcium release (%)

Without PMT

With PMT

No indomethacin Indomethacin

3.3 ± 0.6 4.1 + 0.3

31.9 ± 2.4 13.4 + 1.6**

No hydrocortisone Hydrocortisone

3.3 ± 0.3 4.3 ± 0.5

No flurbiprofen Flurbiprofen

2.0 ± 0.3 0.5 ± 0.4

% Inhibition of calcium release

P~~~~~l-Glucuronidase release (mU/half calvarium)

% Inhibition of glucuronidase release

Without PMT

With PMT

67.5

0.120 ± 0.016 0.104 ± 0.008

0.496 ± 0.030 0.409 + 0.021

18.9

32.1 ± 1.3 8.7 ± 1.7**

84.7

0.138 ± 0.021 0.127 ± 0.014

0.493 + 0.025 0.348 ± 0.076

37.7

26.3 ± 0.9 12.5 ± 1.2***

51.6

0.157 ± 0.010 0.128 ± 0.014

0.517 ± 0.042 0.406 + 0.051*

22.8

a The calvaria were treated in a manner similar to that described in footnote a of Table 1, except that they were incubated for 72 h, instead of 48 h. An inhibitor of prostaglandin synthesis, indomethacin, hydrocortisone, or flurbiprofen (10-6 M) was added for 72 h of incubation. Each value for the release of calcium and of ,B-glucuronidase is given for five half calvaria as mean ± standard error of the mean, and inhibition was calculated as a percentage of the value for the group not treated with a prostaglandin synthesis inhibitor. Values significantly different from the value for the group not treated with a prostaglandin synthesis inhibitor (P < 0.05 [*], P < 0.005 [**], and P < 0.001 [***]) are indicated.

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FIG. 4. Effect of PMT on bone resorption of murine ulnae and radii. After preincubation for 24 h, the ulnae and radii were cultured for 10 days in the presence or absence of increasing amounts of PMT. The release of 45Ca is given as a percentage (mean ± standard error of the mean [SEM]) of total calcium (bone and medium). Values significantly different from that of the control (P < 0.05 [*] and P < 0.005 [**]) are indicated.

In conclusion, we showed that purified PMT induces bone resorption in vitro. Part of this effect is prostaglandin mediated. Whether the protein acts only on bone cells or whether it might induce bone resorption indirectly by stimulating other cells of the nasal cavity is not known. ACKNOWLEDGMENTS We thank S. Palacio for excellent technical assistance and G. Indermuhle and B. Meier for typing the manuscript. This work was supported by the Swiss National Science Foundation (grant 3.894-0.88 SR) and the Danish Governmental Biotechnical Research Programme.

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