Acta path. microbiol. scand. Sect. B, 85: 381-387, 1977
RABBIT POLYMORPHONUCLEAR LEUKOCYTE MIGRATION IN YZYO IN RESPONSE TO LIPOPOLYSACCHARIDES FROM BACTEROZDES, FUSOBACTERIUM AND YEZLLONELLA KJELL SVEEN The Cade Institute, Department of Microbiology, Laboratory for Oral Microbiology, University of Bergen, Bergen, Norway
Sveen, K. Rabbit polymorphonuclear leukocyte migration in vivo in response to lipopolysaccharides from Bactcroidcs, Fusobactcrium and Vcillonclla. Acta path. rnicrobiol. scand. Sect. B, 85, 381-387, 1977. Subcutaneously implanted chambers in rabbits were used for testing the migration of polymorphonuclear leukocytes i n response to injected LF'S isolated from strains of Bactcroidcs, Fusobactsrium and Vcillonclla. A Salmonella LF'S was used as reference endotoxin. No difference in chemotactic activity between the Vcillonclla LPS and LPS from Salmonella was found. Fusobactcrium LPS showed insignificantly lower chemotactic capacity than the Salmonella LPS. The Bactcroidcs LPS were all significantly less chemotactic than the reference endotoxin. An insignificant correlation between the amount of exudate aspirated from the chambers 5 h after injection of the different LPS preparations and the number of leukocytes per pl of exudate was found. c
Key words: Bactcroidcs; Fusobactcrium; Vcillonclla; lipopolysaccharides; leukocyte chemotaxis; wound chambers.
K. Sveen, Mikrobiologisk avdeling, MFH-bygget, N-5016 Haukeland sykehus, Norway.
Received 20.xii.76
Accepted 9.vii.77
Initiation of inflammation through accumulation of polymorphonuclear leukocytes (PMNs) at the injection site is a characteristic feature following administration into animals of lipopolysaccharides (LPS) isolated from Gram-negative bacteria. This has, therefore, made LPS (endotoxin) a valuable tool in studies of the inflammatory response. The production in rabbits of primary skin inflammation and the local and generalized Shwartzman reaction of LPS isolated from
Veillonella alcalescens, Veillonella parvula, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus has been reported (9). It has also been demonstrated that LPS from these microorganisms, when incubated in guinea pig or rabbit serum, elaborate factors chemotactic for rabbit PMNs in vitro (8). The present paper deals with the migration of PMNs in vivo, using wound chambers (10). LPS from the same bacterial organisms as mentioned above was used. It was of
381
particular interest to examine if the Bacteroides LPS, which in in vitro investigations showed a very low activity, induced PMN migration in vivo, and if so, to what extent, when compared with LPS from other anaerobic microorganisms and from Salmonella. MATERIALS AND METHODS Microorganisms The strains used for isolation of LPS were: Bactaroides fragilis subspecies fragilis strains NCTC 9343 and E 323, Bacteroides melaninogenicus subspecies intermedius strain BlO, FusobaFterium nucleatum strains F1 and Fev 1, Veillonella alca!escens strain Ve5 and Veillonella parvula strain Ve9. Details concerning their isolation and cultivation have been reported ( 9 ) . LPS was isolated from whole or disintegrated cells (9) by extraction with phenol-water (12), and purified by ultracentrifugation and treatment with ribonuclease and deoxyribonuclease ( 9 ) . LPS from Salmonella enteritidis S-795 (reference endotoxin) was kindly provided by K . C. Milner (Rocky Mountain Laboratory, Hamilton, Montana, USA). The chemical composition of the LPS preparations and their electronmicroscopical appearance have been reported ( 9 ) . Measurement of Leukocyte Migration Five sterile wound chambers were implanted subcutaneously on each lateral side of six-monthsold New Zealand White rabbits as described earlier ( 10). Six days after implantation, the exudate formed was aspirated and immediately chilled to 0" C. LPS and control solutions were then injected into the chambers as described in Experiments and Results. The amounts of exudate collected from each chamber before and 5 h after injection of LPS and control solutions were measured and 25 pl of it immediately transfernd to 475 pl of methylene blue to determine the number of leukocytes by counting in a Burker chamber. For differential counts, smears of the exudate prepared on glass slides were stained with May-GriinwaldGiemsa (Pappenheim staining). Stock solutions of LPS containing one mg per ml, were prepared in sterile isotonic saline (0.85 per cent). All stock solutions were treated ultrasonically (MSE/Mullard, 60 W, 20 kc/s) a t 0" C for 2 min. If necessary the p H was adjusted to 7.2 0.2 using triethylamine.
*
Statistical Methods Standard deviation was calculated as previously reported (10). The Wilcoxon rank test for two samples (two-tailed) (3) was used for the evaluation of the differences in chemotactic activities
382
exerted by LPS-S-795 and the other endotoxins. The same test was also used for calculating the statistical significance of differences in the responses of rabbits to the test solutions. For the evaluation of the correlation between amount of exudate and number of cells per pl of exudate, the Spearman coefficient of rank correlation was used (3). EXPERIMENTS AND RESULTS
I n preliminary experiments two-fold dilutions of the LPS preparations were tested. When LPS was applied up to a given amount to the granulation tissue within the chambers, a typical dose-response relationship was seen. This was characteristic for each of the LPS preparations and varied from 25-200 pg. The dose levels in the ascending part of the dose-response curves varied according to the high or low chemotactic activity exerted by the various LPS, as also has been reported previously ( 10). When the dose-response curves of the different preparations were compared, 12.5 pg of the LPS was found to lie within the ascending part of all dose-response curves and was, as previously discussed ( 8 ) , chosen for the comparison of the chemotactic activity of the LPS preparations from anaerobic organisms with that exerted by LPS-s-795. LPS in doses of 12.5 pg in 0.4 ml of isotonic saline from each of the eight different LPS preparations were injected into eight chambers on each rabbit. In the remaining two chambers 12.5 pg of glycogen (E. Merck AG, Darmstadt, W-Germany) in 0.4 ml of saline and 0.4 ml of saline alone were injected as controls, respectively. Glycogen was included since LPS-NCTC 9343 and LPSB10 contain large amounts of neutral sugars (9). The sites of the implanted chambers were numbered, and the endotoxin preparations or controls were not injected into the same chamber more than once. The comparative examinations consisted of measurements of the volume of exudate in the chambers before and 5 h after injection of LPS, the number of leukocytes per pl of exudate, and the total ,number of leukocytes within the chamber. T o examine if any biological variance
3
Cells x 10 12 11 10
-
6 5 -
44
T
4 3 -
?1 0
S-795
Ve5
Ve9
F1
Fevl B10
NCTC E323 9343 Sal
Gly
Fig. 1. PMN accumulation in wound chambers induced by lipopolysaccharides isolated from strains of Vcilloncllu, Fusobactcn'urn and Bucteroidcs. For explanation of coded lipopolysaccharides used, see Materials and Methods. Doses of 12.5 p g of each LPS preparation and of glycogen was tested. Each column represents the mean number of cells per pl of wound fluid f standard deviation (vertical bar) from 7 chambers 5 h after injection of test suspensions. Sal = saline, Gly = glycogen. Comparisons are made to LPS-S-795. For statistical analysis the Wilcoxon two-sample (two-tailed) test was used. Values of p 0.05 were accepted as statistically significant. + p 0.05, ++ p 0.02, +++ p 0.005.
>
between the animals could be found, a comparison of the total number of cells in the contents of fluid in all wound chambers in the different rabbits was included. The volume of exudate aspirated from the different chambers at time zero, i.e. six days after the implantation, ranged from 337 -+127.1 to 494.2 f 132.1 pl. The volume of exudate aspirated at time zero from the chambers to be inoculated with LPS-S-795 (337.8 f 127.1 pl), was compared to the volume of exudate in each of the other test chambers (356.6 f 120.4 to 494.2 132.1 1.1). The difference was not statistically significant. The volume of fluid aspirated from the chambers 5 h after injection of the test suspensions, which ranged from 260.7 & 68.1 to 390.0 f 106.3 pl, was not different from that from the chambers injected with the reference endotoxin which measured 295.7 19.1
*
0.1). At time zero there was no difference regarding the number of leukocytes per pl of exudate in the chambers to be inoculated with different test substances (ranging from 408.5 & 92.2 to 457.1 76.1 cells) when compared with those to be injected with LPS-S-795 (392.8 f 109.6 cells) ( p > 0.05). At the end of the experiment (Fig. l ) , no difference in the number of leukocytes per pl of exudate was found when the Veillonella LPS and LPS-F 1 induced wound chamber fluid was compared to that induced by the reference endotoxin ( p > 0.05). The other endotoxins induced less influx of PMNs than
383
* 1fi /j iljfl
5 Cells x 10 3.0
1.o 0.5 0 S795 Ve5
Ve9
F1
Fevl 810
NCTC E323 9343 Sal
Gly
8
Cells x 1
1.5
0.5
Ij 1 Ve5
5-795
Ve9
B
i
ill3 6 Dc
Fevl B10
+++
E323 9343
+++
a
rfi
Sal
G'Y
Fig. 2. Total number of leukocytes in the chambers 6 days after implantation ( A ) and 5 h after injection of bacterial lipopolysaccharides (B) For explanation of coded test suspensions used, comparison, statistical analysis and probability, see legend to Fig. 1. + p 0.05, ++ p 0.05, +++ p 0.005.
.
did LPS-S-795. The difference in number of leukocytes in the LPS-Fev 1 and NCTC 9343 induced chamber fluid was at the two per cent level. Wound fluid from chambers injected with LPS-B10 and LPS-E 323 as well as with glycogen, showed significant leukocyte counts that were only about one third, one fourth and one eleventh of that induced by LPS-S-795 ( p < 0.005). The activity of LPS-NCTC 9343 was significantly higher than glycogen (p < 0.005). LPS-B10 was also more active than glycogen, but less pronounced than LPS-NCTC 9343 was (p < 0.01). As shown in Fig. 2, there was no statistically significant difference in the total number of leukocytes accumulated in the different test chambers at time zero; the mean 384
>
calculated in all the wound chambers in the individual rabbits a t time zero and at the end of the experimental period. Higher numbers of cells were found in the wound fluid in the chambers of rabbits E and F, both before and after the injection of the different LPS and controls. The higher number of cells in the wound chamber fluid of these two animals at the end of the experimental period was
When LPS isolated from strains of Bacteroides, Fusobacterium and Veillonella is applied on a newly formed granulation tissue within chambers subcutaneously implanted in rabbits, this results in an accumulation of polymorphonuclear leukocytes. The in vivo chamber method measures a more complex phenomenon than the in nitro Boyden chamber method does. Thus factors like capillary flow, capillary permeability, mobilization of PMNs from blood depots as well as the rate of PMN production ought to be considered. However, pilot studies concerning fractionations of the wound chamber fluid on Sephadex columns suggest that LPS in vivo as well as in uitro promote chemotaxis through activation of the complement system. Preliminary experiments showed that the elaboration of chemotactic mediators also seem to depend on the physical state of the LPS, since sonical treatment enhanced the attraction of PMNs. This is in accordance with the increased capacity of LPS, when sonicated, to produce skin inflammatory reactions in rabbits (10) and to attract PMNs in vitro (8). The results from this study agree well with the in vitro studies using the modified Boyden chamber device (8). Thus, the relative migratory activity induced by the various LPS showed the same pattern in the in vitro as 385
well as in the in uiuo experiments. This finding indicates that PMN migration in uiuo also is due to chemotaxis. Thus the cheme tactic effect of the Veillonella LPS, either expressed as leukocytes per pl of exudate (cf. Fig. 1) or as number of cells per wound chamber (cf. Fig. 2 ) , was comparable to that of the Salmonella, LPS, whereas the Fusobacterium LPS either was similar or lower in activity. The present study also shows that the Bacteroides LPS possess ,low chemotactic effect on leukocytes when compared to the other LPS and to the Salmonellq LPS. As discussed in a previous paper (8), difference in macromolecular composition may account for the low chemotactic activity of Bucteroides LPS. The Veillonella, LPS and the Fusobqcterium LPS have a molecular composition comparable to that of the Salmonella and this may thus explain their high chemotactic activity The direct or indirect effect of LPS when applied on newly formed granulation tissue, is an efflux of cells as well as a n exudation of fluid, probably due to a n increased permeability of the vessels of this tissue. The correlation between the amount of wound chamber fluid induced by the different LPS preparations and the number of leukocytes per 11.1 of the fluid, however, was insignificant ( p > 0.1). This indicates that a n LPS with a high chemotactic activity on PMNs not necessarily is tantamount to a large amount of exudate formed. A significant variance between the rabbits in the response to the different LPS and controls was found (cf. Fig. 3). I n the model used, however, this does not influence the comparison of chemotactic activity exerted by the different LPS, since they all were tested simultaneously in the same rabbits, The in uiuo chamber method has several advantages over the modified Boyden chamber method: the migration of PMNs as well as MNs may be studied and compared, information about the vascular permeability and the formation of exudate in connection with the inflammatory response may be gained during a defined period of time, and the
.
386
exudate may be subjected to chemical and biological examinations. Chemotaxis physiologically occurs as part of an inflammatory response. A possible connection between chemotaxis and gingivitis has been postulated by Tempe1 et al. (1970). PMNs are thus constantly present both in the gingival crevices and within the junctional epi,thelium of healthy as well as ChronicalIy inflamed gingiva ( 1,2,6). A gradual increase in the number of PMNs within the gingival crevices during the progress of gingivitis has been observed (2). It has also been shown that bacteria per se are chemotactic (4) and that oral bacteria produce factors directly chemotactic for PMNs in uitro ( 1 1 ) . Furthermore, human dental plaque material has been found to possess chemotactic activity for neutrophil leukocytes in uiuo ( 5 ) . Since endotoxin may be liberated from disintegrating Gram-negative bacteria of the human gingival plaque and the quantity of endotoxin in the gingival exudate can be determined ( 7 ) , it seems likely that LPS, by interaction with factors present in the plasma, may contribute to the destruction of tissue in patients suffering from peridontitis. REFERENCES 1. Attstriim, R.: Presence of leukocytes in crevices of healthy and chronically inflamed gingivae. J. periodont. Res. 5: 42-47, 1970. 2. AttstrBm, R . & Egclbcrg, J.: Presence of leukocytes within the gingival crevices during developing gingivitis in dogs. J. periodont. Res. 6: 110-114, 1971. 3. Documcnta Gcigy Scientific Tables. Diem, K . (Ed.). 7. ed. J. R. Geigy S. A., Base1 1975, p. 157-159, 181, 192-193. 4. Kcllcr, H . U. & Sorkin, E.: Studies on chemotaxis. V. On the chemotactic effect of bacteria. Int. Arch. Allergy 31: 505-517, 1967. 5. Lindhe, J . & Hclldln, L.: Neutrophil chemotactic activity elaborated by human dental plaque. J. periodont. Res. 7: 297-303, 1972. 6 . Schroedcr, H . E.: Transmigration and infiltration of leucocytes in human junctional epithelium. Helv. odont. Acta 17: 6-18, 1973. 7. Simon, B. I., Coldman, H . M., Ruben, M . P . & Baker, E.: The role of endotoxin in periodontal disease. I. A reproducible, quantitative
method for determining the amount of endotoxin in human gingival exudate. J. Periodont. 40: 695-701, 1969. 8. Svecn, K.: Rabbit polymorphonuclear leukocyte migration in vitro in response to lipopolysaccharides from Bactcroidcs, Fusobacterium and Veillonella. Acta path. microbiol. scand. Sect. B, 85: 374-380, 1977. 9. Svecn, K.: The capacity of lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella to produce skin inflammation and the local and generalized Shwartzman reaction in rabbits. J. periodont. Res. 12: 340-350, 1977. 10. Sveen, K . & Hofstad, T . : Use of preformed
cavities in rabbits for the quantitation of leukocyte chemotaxis caused by bacterial lipopolysaccharides. Acta path. microbiol. scand. Sect. B, 84: 252-258, 1976. 11. Tempel, T . R., Snyderman, R., Jordan, H.V . & Mergenhagen, S . E.: Factors from saliva and oral bacteria, chemotactic for polymorphonuclear leukocytes: their possible role in gingival inflammation. J. Periodont. 41: 7179, 1970. 12. Westphal, O., Liideritx, 0 . & Bister, F.: Uber die Extraktion von Bakterien mit Phenol/ Wasser. Z. Naturforsch. 7B: 148-155, 1952.
387
RABBIT POLYMORPHONUCLEAR LEUKOCYTE MIGRATION IN YZYO IN RESPONSE TO LIPOPOLYSACCHARIDES FROM BACTEROZDES, FUSOBACTERIUM AND YEZLLONELLA KJELL SVEEN The Cade Institute, Department of Microbiology, Laboratory for Oral Microbiology, University of Bergen, Bergen, Norway
Sveen, K. Rabbit polymorphonuclear leukocyte migration in vivo in response to lipopolysaccharides from Bactcroidcs, Fusobactcrium and Vcillonclla. Acta path. rnicrobiol. scand. Sect. B, 85, 381-387, 1977. Subcutaneously implanted chambers in rabbits were used for testing the migration of polymorphonuclear leukocytes i n response to injected LF'S isolated from strains of Bactcroidcs, Fusobactsrium and Vcillonclla. A Salmonella LF'S was used as reference endotoxin. No difference in chemotactic activity between the Vcillonclla LPS and LPS from Salmonella was found. Fusobactcrium LPS showed insignificantly lower chemotactic capacity than the Salmonella LPS. The Bactcroidcs LPS were all significantly less chemotactic than the reference endotoxin. An insignificant correlation between the amount of exudate aspirated from the chambers 5 h after injection of the different LPS preparations and the number of leukocytes per pl of exudate was found. c
Key words: Bactcroidcs; Fusobactcrium; Vcillonclla; lipopolysaccharides; leukocyte chemotaxis; wound chambers.
K. Sveen, Mikrobiologisk avdeling, MFH-bygget, N-5016 Haukeland sykehus, Norway.
Received 20.xii.76
Accepted 9.vii.77
Initiation of inflammation through accumulation of polymorphonuclear leukocytes (PMNs) at the injection site is a characteristic feature following administration into animals of lipopolysaccharides (LPS) isolated from Gram-negative bacteria. This has, therefore, made LPS (endotoxin) a valuable tool in studies of the inflammatory response. The production in rabbits of primary skin inflammation and the local and generalized Shwartzman reaction of LPS isolated from
Veillonella alcalescens, Veillonella parvula, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus has been reported (9). It has also been demonstrated that LPS from these microorganisms, when incubated in guinea pig or rabbit serum, elaborate factors chemotactic for rabbit PMNs in vitro (8). The present paper deals with the migration of PMNs in vivo, using wound chambers (10). LPS from the same bacterial organisms as mentioned above was used. It was of
381
particular interest to examine if the Bacteroides LPS, which in in vitro investigations showed a very low activity, induced PMN migration in vivo, and if so, to what extent, when compared with LPS from other anaerobic microorganisms and from Salmonella. MATERIALS AND METHODS Microorganisms The strains used for isolation of LPS were: Bactaroides fragilis subspecies fragilis strains NCTC 9343 and E 323, Bacteroides melaninogenicus subspecies intermedius strain BlO, FusobaFterium nucleatum strains F1 and Fev 1, Veillonella alca!escens strain Ve5 and Veillonella parvula strain Ve9. Details concerning their isolation and cultivation have been reported ( 9 ) . LPS was isolated from whole or disintegrated cells (9) by extraction with phenol-water (12), and purified by ultracentrifugation and treatment with ribonuclease and deoxyribonuclease ( 9 ) . LPS from Salmonella enteritidis S-795 (reference endotoxin) was kindly provided by K . C. Milner (Rocky Mountain Laboratory, Hamilton, Montana, USA). The chemical composition of the LPS preparations and their electronmicroscopical appearance have been reported ( 9 ) . Measurement of Leukocyte Migration Five sterile wound chambers were implanted subcutaneously on each lateral side of six-monthsold New Zealand White rabbits as described earlier ( 10). Six days after implantation, the exudate formed was aspirated and immediately chilled to 0" C. LPS and control solutions were then injected into the chambers as described in Experiments and Results. The amounts of exudate collected from each chamber before and 5 h after injection of LPS and control solutions were measured and 25 pl of it immediately transfernd to 475 pl of methylene blue to determine the number of leukocytes by counting in a Burker chamber. For differential counts, smears of the exudate prepared on glass slides were stained with May-GriinwaldGiemsa (Pappenheim staining). Stock solutions of LPS containing one mg per ml, were prepared in sterile isotonic saline (0.85 per cent). All stock solutions were treated ultrasonically (MSE/Mullard, 60 W, 20 kc/s) a t 0" C for 2 min. If necessary the p H was adjusted to 7.2 0.2 using triethylamine.
*
Statistical Methods Standard deviation was calculated as previously reported (10). The Wilcoxon rank test for two samples (two-tailed) (3) was used for the evaluation of the differences in chemotactic activities
382
exerted by LPS-S-795 and the other endotoxins. The same test was also used for calculating the statistical significance of differences in the responses of rabbits to the test solutions. For the evaluation of the correlation between amount of exudate and number of cells per pl of exudate, the Spearman coefficient of rank correlation was used (3). EXPERIMENTS AND RESULTS
I n preliminary experiments two-fold dilutions of the LPS preparations were tested. When LPS was applied up to a given amount to the granulation tissue within the chambers, a typical dose-response relationship was seen. This was characteristic for each of the LPS preparations and varied from 25-200 pg. The dose levels in the ascending part of the dose-response curves varied according to the high or low chemotactic activity exerted by the various LPS, as also has been reported previously ( 10). When the dose-response curves of the different preparations were compared, 12.5 pg of the LPS was found to lie within the ascending part of all dose-response curves and was, as previously discussed ( 8 ) , chosen for the comparison of the chemotactic activity of the LPS preparations from anaerobic organisms with that exerted by LPS-s-795. LPS in doses of 12.5 pg in 0.4 ml of isotonic saline from each of the eight different LPS preparations were injected into eight chambers on each rabbit. In the remaining two chambers 12.5 pg of glycogen (E. Merck AG, Darmstadt, W-Germany) in 0.4 ml of saline and 0.4 ml of saline alone were injected as controls, respectively. Glycogen was included since LPS-NCTC 9343 and LPSB10 contain large amounts of neutral sugars (9). The sites of the implanted chambers were numbered, and the endotoxin preparations or controls were not injected into the same chamber more than once. The comparative examinations consisted of measurements of the volume of exudate in the chambers before and 5 h after injection of LPS, the number of leukocytes per pl of exudate, and the total ,number of leukocytes within the chamber. T o examine if any biological variance
3
Cells x 10 12 11 10
-
6 5 -
44
T
4 3 -
?1 0
S-795
Ve5
Ve9
F1
Fevl B10
NCTC E323 9343 Sal
Gly
Fig. 1. PMN accumulation in wound chambers induced by lipopolysaccharides isolated from strains of Vcilloncllu, Fusobactcn'urn and Bucteroidcs. For explanation of coded lipopolysaccharides used, see Materials and Methods. Doses of 12.5 p g of each LPS preparation and of glycogen was tested. Each column represents the mean number of cells per pl of wound fluid f standard deviation (vertical bar) from 7 chambers 5 h after injection of test suspensions. Sal = saline, Gly = glycogen. Comparisons are made to LPS-S-795. For statistical analysis the Wilcoxon two-sample (two-tailed) test was used. Values of p 0.05 were accepted as statistically significant. + p 0.05, ++ p 0.02, +++ p 0.005.
>
between the animals could be found, a comparison of the total number of cells in the contents of fluid in all wound chambers in the different rabbits was included. The volume of exudate aspirated from the different chambers at time zero, i.e. six days after the implantation, ranged from 337 -+127.1 to 494.2 f 132.1 pl. The volume of exudate aspirated at time zero from the chambers to be inoculated with LPS-S-795 (337.8 f 127.1 pl), was compared to the volume of exudate in each of the other test chambers (356.6 f 120.4 to 494.2 132.1 1.1). The difference was not statistically significant. The volume of fluid aspirated from the chambers 5 h after injection of the test suspensions, which ranged from 260.7 & 68.1 to 390.0 f 106.3 pl, was not different from that from the chambers injected with the reference endotoxin which measured 295.7 19.1
*
0.1). At time zero there was no difference regarding the number of leukocytes per pl of exudate in the chambers to be inoculated with different test substances (ranging from 408.5 & 92.2 to 457.1 76.1 cells) when compared with those to be injected with LPS-S-795 (392.8 f 109.6 cells) ( p > 0.05). At the end of the experiment (Fig. l ) , no difference in the number of leukocytes per pl of exudate was found when the Veillonella LPS and LPS-F 1 induced wound chamber fluid was compared to that induced by the reference endotoxin ( p > 0.05). The other endotoxins induced less influx of PMNs than
383
* 1fi /j iljfl
5 Cells x 10 3.0
1.o 0.5 0 S795 Ve5
Ve9
F1
Fevl 810
NCTC E323 9343 Sal
Gly
8
Cells x 1
1.5
0.5
Ij 1 Ve5
5-795
Ve9
B
i
ill3 6 Dc
Fevl B10
+++
E323 9343
+++
a
rfi
Sal
G'Y
Fig. 2. Total number of leukocytes in the chambers 6 days after implantation ( A ) and 5 h after injection of bacterial lipopolysaccharides (B) For explanation of coded test suspensions used, comparison, statistical analysis and probability, see legend to Fig. 1. + p 0.05, ++ p 0.05, +++ p 0.005.
.
did LPS-S-795. The difference in number of leukocytes in the LPS-Fev 1 and NCTC 9343 induced chamber fluid was at the two per cent level. Wound fluid from chambers injected with LPS-B10 and LPS-E 323 as well as with glycogen, showed significant leukocyte counts that were only about one third, one fourth and one eleventh of that induced by LPS-S-795 ( p < 0.005). The activity of LPS-NCTC 9343 was significantly higher than glycogen (p < 0.005). LPS-B10 was also more active than glycogen, but less pronounced than LPS-NCTC 9343 was (p < 0.01). As shown in Fig. 2, there was no statistically significant difference in the total number of leukocytes accumulated in the different test chambers at time zero; the mean 384
>
calculated in all the wound chambers in the individual rabbits a t time zero and at the end of the experimental period. Higher numbers of cells were found in the wound fluid in the chambers of rabbits E and F, both before and after the injection of the different LPS and controls. The higher number of cells in the wound chamber fluid of these two animals at the end of the experimental period was
When LPS isolated from strains of Bacteroides, Fusobacterium and Veillonella is applied on a newly formed granulation tissue within chambers subcutaneously implanted in rabbits, this results in an accumulation of polymorphonuclear leukocytes. The in vivo chamber method measures a more complex phenomenon than the in nitro Boyden chamber method does. Thus factors like capillary flow, capillary permeability, mobilization of PMNs from blood depots as well as the rate of PMN production ought to be considered. However, pilot studies concerning fractionations of the wound chamber fluid on Sephadex columns suggest that LPS in vivo as well as in uitro promote chemotaxis through activation of the complement system. Preliminary experiments showed that the elaboration of chemotactic mediators also seem to depend on the physical state of the LPS, since sonical treatment enhanced the attraction of PMNs. This is in accordance with the increased capacity of LPS, when sonicated, to produce skin inflammatory reactions in rabbits (10) and to attract PMNs in vitro (8). The results from this study agree well with the in vitro studies using the modified Boyden chamber device (8). Thus, the relative migratory activity induced by the various LPS showed the same pattern in the in vitro as 385
well as in the in uiuo experiments. This finding indicates that PMN migration in uiuo also is due to chemotaxis. Thus the cheme tactic effect of the Veillonella LPS, either expressed as leukocytes per pl of exudate (cf. Fig. 1) or as number of cells per wound chamber (cf. Fig. 2 ) , was comparable to that of the Salmonella, LPS, whereas the Fusobacterium LPS either was similar or lower in activity. The present study also shows that the Bacteroides LPS possess ,low chemotactic effect on leukocytes when compared to the other LPS and to the Salmonellq LPS. As discussed in a previous paper (8), difference in macromolecular composition may account for the low chemotactic activity of Bucteroides LPS. The Veillonella, LPS and the Fusobqcterium LPS have a molecular composition comparable to that of the Salmonella and this may thus explain their high chemotactic activity The direct or indirect effect of LPS when applied on newly formed granulation tissue, is an efflux of cells as well as a n exudation of fluid, probably due to a n increased permeability of the vessels of this tissue. The correlation between the amount of wound chamber fluid induced by the different LPS preparations and the number of leukocytes per 11.1 of the fluid, however, was insignificant ( p > 0.1). This indicates that a n LPS with a high chemotactic activity on PMNs not necessarily is tantamount to a large amount of exudate formed. A significant variance between the rabbits in the response to the different LPS and controls was found (cf. Fig. 3). I n the model used, however, this does not influence the comparison of chemotactic activity exerted by the different LPS, since they all were tested simultaneously in the same rabbits, The in uiuo chamber method has several advantages over the modified Boyden chamber method: the migration of PMNs as well as MNs may be studied and compared, information about the vascular permeability and the formation of exudate in connection with the inflammatory response may be gained during a defined period of time, and the
.
386
exudate may be subjected to chemical and biological examinations. Chemotaxis physiologically occurs as part of an inflammatory response. A possible connection between chemotaxis and gingivitis has been postulated by Tempe1 et al. (1970). PMNs are thus constantly present both in the gingival crevices and within the junctional epi,thelium of healthy as well as ChronicalIy inflamed gingiva ( 1,2,6). A gradual increase in the number of PMNs within the gingival crevices during the progress of gingivitis has been observed (2). It has also been shown that bacteria per se are chemotactic (4) and that oral bacteria produce factors directly chemotactic for PMNs in uitro ( 1 1 ) . Furthermore, human dental plaque material has been found to possess chemotactic activity for neutrophil leukocytes in uiuo ( 5 ) . Since endotoxin may be liberated from disintegrating Gram-negative bacteria of the human gingival plaque and the quantity of endotoxin in the gingival exudate can be determined ( 7 ) , it seems likely that LPS, by interaction with factors present in the plasma, may contribute to the destruction of tissue in patients suffering from peridontitis. REFERENCES 1. Attstriim, R.: Presence of leukocytes in crevices of healthy and chronically inflamed gingivae. J. periodont. Res. 5: 42-47, 1970. 2. AttstrBm, R . & Egclbcrg, J.: Presence of leukocytes within the gingival crevices during developing gingivitis in dogs. J. periodont. Res. 6: 110-114, 1971. 3. Documcnta Gcigy Scientific Tables. Diem, K . (Ed.). 7. ed. J. R. Geigy S. A., Base1 1975, p. 157-159, 181, 192-193. 4. Kcllcr, H . U. & Sorkin, E.: Studies on chemotaxis. V. On the chemotactic effect of bacteria. Int. Arch. Allergy 31: 505-517, 1967. 5. Lindhe, J . & Hclldln, L.: Neutrophil chemotactic activity elaborated by human dental plaque. J. periodont. Res. 7: 297-303, 1972. 6 . Schroedcr, H . E.: Transmigration and infiltration of leucocytes in human junctional epithelium. Helv. odont. Acta 17: 6-18, 1973. 7. Simon, B. I., Coldman, H . M., Ruben, M . P . & Baker, E.: The role of endotoxin in periodontal disease. I. A reproducible, quantitative
method for determining the amount of endotoxin in human gingival exudate. J. Periodont. 40: 695-701, 1969. 8. Svecn, K.: Rabbit polymorphonuclear leukocyte migration in vitro in response to lipopolysaccharides from Bactcroidcs, Fusobacterium and Veillonella. Acta path. microbiol. scand. Sect. B, 85: 374-380, 1977. 9. Svecn, K.: The capacity of lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella to produce skin inflammation and the local and generalized Shwartzman reaction in rabbits. J. periodont. Res. 12: 340-350, 1977. 10. Sveen, K . & Hofstad, T . : Use of preformed
cavities in rabbits for the quantitation of leukocyte chemotaxis caused by bacterial lipopolysaccharides. Acta path. microbiol. scand. Sect. B, 84: 252-258, 1976. 11. Tempel, T . R., Snyderman, R., Jordan, H.V . & Mergenhagen, S . E.: Factors from saliva and oral bacteria, chemotactic for polymorphonuclear leukocytes: their possible role in gingival inflammation. J. Periodont. 41: 7179, 1970. 12. Westphal, O., Liideritx, 0 . & Bister, F.: Uber die Extraktion von Bakterien mit Phenol/ Wasser. Z. Naturforsch. 7B: 148-155, 1952.
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