Acta path. microbiol. scand. Sect. B. 86: 93-100. 1978.

THE IMPORTANCE OF C5 AND THE ROLE OF THE ALTERNATIVE COMPLEMENT PATHWAY IN LEUKOCYTE CHEMOTAXIS INDUCED IN VIVO AND IN VITRO

BY BA CTEROIDES FRA GILIS LIPOPOLYSACCHARIDE WELL SVEEN The Gade Institute. Department of Microbiology. Laboratory of Oral Microbiology. University of Bergen. Bergen. Norway

Sveen, K. The importance of C5 and the role of the alternative complement pathway in leukocyte chemotaxis induced in vivo and in virro by Bucleroides .fragi/is lipopolysaccharide. Acta path. microbiol. a n d . Sect. B. 86: 93-100, 1978. Chambers implanted subcutaneously in C5 normal (C5 N) and C5 deficient (CS D) mice were used to examine the migration of polymorphonuclear leukocytes (PMNs) into the wound chamber fluid in response to injected Bucteroides .frugi/is lipopolysaccharide (LPS). The difference in PMN migration was highly significant between the two mouse strains. the C 5 D mice showing no initial, but a low, delayed migration. The results from the study indicated that chemotaxis plays a major role in the accumulation of PMNs in the acute inflammatory response. Intraperitoneal endotoxin stimulation also showed a significantly lower total number of leukocytes in the exudate from CS D mice as well as a delayed migration of cells compared to C5 N mice. No leukotactic mediators were elaborated in C5 D serum or exudate upon incubation with LPS when tested in a modified Boyden chamber. However, endotoxininduced wound chamber fluid in C5 D mice showed an increasing leukotactic activity at the same time as the acute inflammatory response subsided in C5 N mice. Incubation of B. fragilis LPS in C4 deficient (C4 D)guinea pig serum indicated that the LPS was able to activate complement components to generate split products chemotactic for rabbit PMNs via the alternative complement pathway. Key words: CS: alternative complement pathway: chemotaxis: Bucteroides ,frugi/is: lipopolysaccharide. Kjell Sveen, Mikrobiologisk avdeling. MFH-bygget. N-5016 Haukeland sykehus. Norway.

Received 2 7 x 7 7

Accepted 4.xi.77

The endotoxicity of lipopolysaccharides (LPS) from Bacteroides fragilis has been questioned (7). Nevepheless, investigations carried out in this laboratory have shown that B. fragilis LPS produce primary skin inflammation as well as local and generalized Shwartzman reaction in rabbits (1 2). In addition, the LPS were lethal for mice and chick embryos, pyrogenic in rabbits and caused gelation of Limulus amoebocyte lysate (1 6). In all these tests the endotoxin activity was characteristically low. The B. fragilis LPS, which were chemotactically active in vivo (1 4), also showed a serum-dependent in v i m chemotaxis for polymorphonuclear leukocytes BMNs) (1 3).

Snyderman el al. (10) have shown that complement (C) is implicated in the early accumulation of PMNs in inflammatory exudates produced by LPS. During the elaboration of these inflammatory mediators, which are cleavage products from C formed during the interaction with LPS, there is a pronounced consumption of the terminal C components C3 through C9, but only minimal using up of C1, C4 and C2 (4, 5, 6). The C5 was the main factor for mediation of the chemotactic activity ( I 0, 1 I). The present study was performed to examine the importance of various C components, particularly the C5, in the elaboration of leukotactic mediators upon interaction of B. fragilis LPS. Mice genetically

93

deficient in CS (C5 D) and CS normal mice (CS N), were employed in the experiments. Furthermore, the ability of B. fragilis LPS to activate the alternative complement pathway in the induction of chemotactic factor production was examined using guinea pig serum genetically deficient in C4 (C4 D).

Measurement of Migration of Leukocytes into rhe Peritoneal Cavity Suspensions of LPS-E 323 in amounts of 150 pg in

bers

Complement Source

Teflon@chambers (8) of 22 mm length and with a diameter of 6 mm, were implanted subcutaneously in the mice, one on each lateral side of the body of each animal. The preoperative preparation of the mice and the operative procedure was performed as described earlier (15). Seven days after the implantation. the formed exudate (wound fluid) in the chamber was aspirated, and the volume measured. The exudate was immediately brought to Oo C in an ice-bath, after which 25 pl of exudate were added to 475 p1 of methylene blue, and the number of leukocytes counted using a Biirker chamber. The aspirated exudate was replaced by a suspension of 50 pg of LPS-E 323 in 0.2 ml of sterile isotonic saline into one chamber and the same amount of saline into the other chamber as control. At different time intervals after the application of the test substances, the exudate was aspirated and its volume measured, and the number of leukocytes per wound chamber calculated. The exudate aspirated seven days after the implantation of the chambers was checked for contaminating microorganisms by microscopy of Gram-stained preparations and from anaerobic and aerobic incubation on blood agar plates.

Fresh, pooled serum from genetically C4 D and C4 N guinea pigs, as well as from CS D and CS N mice, was stored at - 2 5 O C in aliquots of one ml. In addition, exudate aspirated from the implanted chambers before and after stimulation with LPS was used. The exudate was centrifuged at 28.000 x g for 20 min at 4O C (Servall RC-2, Sorvall Inc.. Norwalk, COM., USA) and the supernatant fluid frozen in aliquots of one ml. Endotoxin or saline stimulated peritoneal exudate was centrifuged at 28.000 X g and lyophilized. The lyophilized exudate from three mice was redissolved in I ml of distilled water and dialyzed against saline of at least 1000 times their volume using a magnetic mixer at 4O C for 24 h and thereafter lyophilized. Before testing on chemotactic activity, the freeze-dried exudate was dissolved in 0.8 ml of Gey's medium, thus giving a concentration of the exudate ten times higher than at the harvesting.

0.9 ml sterile isotonic saline were injected intraperitoneally (i.p.). At different time intervals thereafter the mice were sacrificed by cervical dislocation, and the peritoneal cavity was exposed by an abdominal incision. The exudate was collected either by rinsing the peritoneal cavity with 0.005 M sodium ethylenediamine tetraacetate (EDTA) in saline (pH 7.2) to block the activation of MATERIALS AND METHODS complement (10) or only saline, both containing 10 I.U. Source and Preparation of LPS of heparin (A/S Apothekernes Laboratorium for SpeciLPS (endotoxin)was isolated from Bacreroidesfragilis alpneparater, Oslo) per ml and immediately brought to ss. fragilis strain Lille E 323 by phenol-water extraction Oo C. The volume of exudate from each mouse was ( I 7) and purified by ultracentrifugation and treatment adjusted to a volume of 2.7 ml. and 25 pl of this with ribonuclease and deoxyribonuclease (12). Stock transferred to 475 pl of methylene blue to determine the suspensions of LPS were made in sterile isotonic saline number of leukocytes per pl of exudate. (0.85 per cent NaC1) for in vlvo assays and in Gey's balanced salt solution (Gey's medium) for in vitro assays Chemotactic Assays and sonicated (MSE-Mullard, 60w, 20 kc/d for one min Polymorphonuclear leukocytes. Polymorphonuclear at Oo C. The pH was adjusted to 7.2 using triethylamine. leukocytes (PMNs) were harvested from the peritoneal cavity of rabbits 8-10 h after i.p. stimulation with 200 Animals ml of a solution containing 0.1 per cent glycogen (E. Mice of both sexes of the CS normal strain NMRJ. Merck AG, Darmstadt. W-Germany) in sterile isotonic weighing 32 f 3 g, and of the strain DBA/2J genetically saline. After isolation of the PMNs by centrifugation at deficient in CS weighing 28 f 2 g were used, the two 225 X g for 5 min (IEC. Universal Model UV), they mouse strains being caged separately. Adult guinea pigs, were resuspended in Gey's medium containing 50 pg normal and genetically deficient in C4 (homozygous) garamycin@(Schering Corporation. Bloomfield. N. J., (supplied by MRC Lab. Animals Centre, Surrey, USA) and 2.5 pg fungizone" (Squibb, Flow LaboratoEngland), were used. Their weights ranged from 400- ries, Irvine. Scotland) per ml and 2 per cent bovine 600 g. and the two strains were housed separately. New serum albumin (BSA) (Armour Pharmaceutical ComZealand White rabbits. 5-6-months-old and weighing pany Ltd., Eastbourne, England). The leukocyte suspen3.5-4.0 kg. were also caged separately. All animals were sion was adjusted to give a final number of approximamaintained on a standard laboratory diet and water ad tely 10' leukocytes per ml. The media used were lib. They were kept at a temperature of 20-22' C and a sterilized before use by passage through a filter with a relative humidity of 45-50. pore size of 0.45 p (Millipore Filter Corp.. Bedford. Mass., USA). Measurement of Leukocyte Migration into Wound Cham-

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Measurement of Chemotactic Activity

Chemotaxis of rabbit PMNs was quantitatively assayed by the Boyden Micropore filter technique ( I ) using a modified Boyden chamber (1 I ) (Neuroprobe. Bethesda. Md., USA). Filters of 3.0 p pore size

(Miflipore Filter Corp.) were used to separate the chamber in two compartments. The bottom compartment, having a volume of 0.2 ml. contained the fluid to be tested for chemotactic activity. The ratio of the complement source to Gey's medium, with or without LPS, was I : I . Aliquots of 1.2 ml of the mixtures or controls were preincubated in a water-bath at 37' C for 30 min and thereafter at 56O C for 30 min. Exudate aspirated from in vivo chambers 5 and 20 h after injection of endotoxin was preincubated at 56' C only before being tested for chemotactiv activity. The volume of 0.4 ml of the leukocyte suspension was spun down on the filter (58 x g for I .S min) in a cytocentrifuge (Shandon Elliot Cytospin SCA-0030). thus giving a total of approximately 4 x lo6 PMNs on the upper surface (13) and the top compartment filled up with 0.4 ml of Gey's medium containing antibiotics and BSA. After 3 h of incubation in humid air at 37' C the filters were removed, stained and cleared. These were then mounted as described previously ( I 3). Chemotactic activity was quantitated by counting the numbers of PMNs. which had migrated completely through the filters. in five highpower fields under light microscopy ( 13).

6 Cells x 10

A

Ii

El&

Statistical Methods

B

For evaluation of differences of chemotactic activities. the Wilcoxon rank test for two samples (two-tailed) was used (3). Values of p>O.OS were not accepted as statistically significant. The Pearson correlation coefficient (3) was used for calculation of the linearity of the time-response curves.

EXPERIMENTS AND RESULTS Seven days after the implantation, the chambers were surrounded by a granulation tissue, buds of which were growing through the perforations of the chamber wall, thus forming a lining of its lumen. The survival at this time was eigth out of ten C5 D and all ten of C5 N mice. The volume of exudate aspirated from the chambers in the C5 N and C5 D mice seven days after implantation (time zero) and 5 h and 20 h after injection of 50 pg LPS-E 323 in 0.2 ml saline or saline alone into the chambers, is shown in Table I .

L

0

20

Hours after inject ion Fig. 1 . Total number of leukocytes in wound chamber

fluid seven days after implantation (0) and five and 20 h after application of 100 pg LPS-E 323 in 0.2 ml saline. or only saline. in CS N (A) and CS D (9) mice. Each column represents the mean number of cells per wound chamber 2 standard deviation (vertical bar) from five chambers except at time zero where the number was ten. 0 endotoxin induced wound fluid, saline induced wound fluid.

TABLE I . Volume (PI)or Exudare in

rile Wound Ciiumbers o f C 5 Normal (CS N ) and C5 Deficient (CS D ) Mice Before (Time Zero) and Jive and 20 h Afrer Injection of LPS-E 323 in Saline. or Saline alone

Before injection Mouse strain

CS N CS D

Five h after injection

20 h after injection

Mean 5s.d.

LPS Mean +- s.d.

Saline Mean 2s.d.

LPS Mean 2 s.d.

Saline Mean 2s.d.

156232 179241

141 t 3 6 175577

147267 198265

161 +-46 116543

150232 I I8 220

Mean and standard deviation (s.d.) are calculated from five chambers. The difference in amount of exudate aspirated from CS N and CS D mice was not significant either before or after the stimulation with LPS or saline.

95

6 Cells x 10 22 20 -

-

16 14 18

12 10

-

-

8 6 -

0 1

3

5 7 10 Hours after injection

20

Fig.2. Kinetics of leukocyte accumulation in the peritoneal cavity of CS N and CS D mice after local injection of SO pg LPS-E 323 in 0.9 ml saline, or saline alone. Each point represents the mean cell count 2 standard deviation (vertical bar) from three mice. There is a time differenceof half an hour between the aspirationof endotoxin and saline induced exudate. -0- CS N mice injected with LPS -0- CS D mice injected with LPS CS N mice injected with saline -0- CS D mice injected with saline

+

Comparison of the amount of exudate from chambers injected with endotoxin suspension to that from chambers containing saline 5 h and 20 h later gave no statistical significancein either of the mouse strains (p>0.05). At time zero no significant difference in the total number of cells per wound chamber was to be observed when C5 N and C5 D mice were compared (p>0.05) (Fig. I). Five h after the application of endotoxin, the total number of cells in the wound chamber fluid from C5 N mice was significantly higher than that from C5 D mice (p

The importance of C5 and the role of the alternative complement pathway in leukocyte chemotaxis induced in vivo and in vitro by Bacteroides fragilis lipopolysaccharide.

Acta path. microbiol. scand. Sect. B. 86: 93-100. 1978. THE IMPORTANCE OF C5 AND THE ROLE OF THE ALTERNATIVE COMPLEMENT PATHWAY IN LEUKOCYTE CHEMOTAX...
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