Summary. The role of granulocytes in the induction of endocarditis with a dextran-producing. Streptococcus sanguis and a dextran-negative mutant of this strain ...
Br. 1. exp. Path. (I984) 65, 257-265
Role of granulocytes in the induction of an experimental endocarditis with a dextran-producing Streptococcus sanguis and its dextran-negative mutant M.J.M. Meddens, J. Thompson, P.C.J. Leijh and R. van Furth
Department of Infectious Diseases, Rijnsburgerweg 1o, University Hospital, 233AA Leiden, The Netherlands
Received for publication I 3 December I983
Summary. The role of granulocytes in the induction of endocarditis with a dextran-producing Streptococcus sanguis and a dextran-negative mutant of this strain was studied. The number of colony-forming units of Streptococcus sanguis needed to colonize the vegetations in 50% of the rabbits (ID50) was significantly lower for the parent strain than for the dextran-negative mutant. However, in granulocytopenic rabbits the ID50s of both strains did not differ measurably. Dextran-negative streptococci were more readily cleared from the circulation than dextran-positive, but in this respect no difference was found between control and granulocytopenic rabbits, which indicates that clearance cannot account for the difference in ID5o between the two strains in the control group. At serum concentrations of 5% and lower, in-vitro granulocytes phagocytosed the dextran-negative streptococci more rapidly than the dextran-positive. The intracellular killing of the streptococci was not influenced by dextran production. This study suggests that an impaired phagocytic removal ofattached bacteria from the vegetational surface can be a factor promoting the induction of endocarditis by dextran-producing streptococci.
Keywords: granulocytes, experimental endocarditis, dextran, Streptococcus sanguis The pathogenesis of bacterial endocarditis is a complex process involving the interaction of several independent factors (Angrist & Oka I963). Several experimental studies suggest that bacterial adherence could be one of these factors. For instance a good correlation was found between the adherence of bacterial strains to isolated strips of heart valve or synthetic analogues of endocardial vegetations in vitro and their ability to cause endocarditis in vivo (Gould et al. 1975; Ramirez-Ronda I978; Scheld et al. I978). For streptococci dextran was found both to increase virulence and to promote adherence. For the induction of a Streptococcus
sanguis endocarditis a significantly lower number of a dextran-producing strain was needed than of its dextran-negative mutant (Pelletier et al. I978; Scheld et al. 1978). Furthermore, treatment of the streptococci with dextranase significantly reduced their ability to induce endocarditis (Scheld et al. 1 9 78). The number of streptococci needed to induce endocarditis in vivo generally showed good correlation with the amount of dextran they produced and their adherence to fibrinplatelet surfaces in vitro (Scheld et al. I978). Although these findings suggest an important role of bacterial adherence in the pathogenesis of endocarditis, they do not rule out 257
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M.J.M. Meddens et al. that other mechanisms contribute to the Micro-organisms. Use was made of a dextrandivergent infectivity of different strains. Such producing Streptococcus sanguis (NCTC a mechanism could be phagocytic removal of 7864) and a dextran-negative mutant of this bacteria from the surface of endocardial strain, kindly provided by Dr Groothuis ofthe vegetations before the micro-organisms National Institute of Public Health (RIV, become covered by fibrin. The present study Bilthoven, The Netherlands). The dextranwas undertaken to find out whether dextran negative streptococcus is a spontaneous facilitates the induction of Streptococcus san- mutant isolated from a culture in Toddguis endocarditis by enhancing attachment Hewitt broth ofthe parent strain that was the of blood-borne micro-organisms to endocar- same as the one used earlier (Van Ginkel et al. dial vegetations or by hampering phagocytic I979; Meddens et al. I982; Thompson et al. removal by granulocytes of attached streptoI976; Thbrig et al. I980). Dextran-positive cocci from the vegetational surface through and dextran-negative streptococci were difan effect on phagocytosis. ferentiated after 48 h of incubation under anaerobic conditions on 5% sucrose trypticase yeast cystein agar on morphological criteria: colonies of dextran-positive streptoMaterials and methods cocci show a bright elevated centre, whereas Experimental design. The study was done in colonies of dextran-negative mutants are flat male New Zealand White rabbits weighing and have a homogeneous appearance. The 2-3 kg. In each experiment, the rabbits were production of dextran was investigated in numbered randomly (Statistics applications both strains with a fractionated ethanol and 1978), and each step was carried out in this acetone/methanol precipitation as described sequence. Non-bacterial thrombotic endo- by Guthoff (I970). The parent strain was carditis was induced by inserting a plastic found to produce large amounts of dextran, catheter via the left carotid artery into the left whereas the mutant streptococcus did not ventricle, according to Durack & Beeson show detectable dextran production after (I972) and Thompson et al (I976). To incubation in Todd-Hewitt medium suppleinduce granulocytopenia, nitrogen mustard mented with 5% sucrose. After six in-vitro (HN2) was administered immediately after passages on 5% sucrose trypticase yeast catheterization. The catheter was left in situ cystein agar no dextran production by the during the experiment. Three days later, i ml mutant strain was demonstrable with the of a suspension of live streptococci was Guthof procedure. After each passage the injected intravenously into a marginal ear identity of the mutant streptococcus was vein. Forty-eight hours later, the rabbits verified by serotyping, which showed that were killed by injection of sodium pentobar- the serotype of the mutant strain remained bital and vegetations were removed asepti- the same as that of the native streptococcus. cally for quantitative bacteriology. For infection of the vegetations and for the phagocytosis and killing experiments, overnight cultures of the native strain and the Induction of granulocytopenia. Nitrogen mus- dextran-negative mutant in Todd-Hewitt tard (HN2, Mustine hydrochloride, ACF, medium were used. To allow the production Chemie Farma B.V., Maarssen, The Nether- of dextran, the medium for the native streplands) was used to induce granulocytopenia tococcus was supplemented with 5% sucas described elsewhere (Meddens et al. rose, which is known to be the only substrate i982). Briefly, io mg HN2 was dissolved in for the production of dextran (Hehre & Neil io ml sterile saline just before use. Each 1946). The cultures each containing about rabbit received 5 mg in a single injection into Io8 colony-forming units (CFU) per ml, were a marginal ear vein. washed three times and diluted to the appro-
Role of granulocytes in experimental endocarditis priate concentration in saline. To maintain optimal condition of the streptococci, the saline was enriched with i% bacteriological peptone (Oxoid) throughout. After each animal passage, the native and mutant streptococcus were checked for dextran production on the basis of the morphological criteria mentioned above.
Quantitative bacteriology. The methods used for the quantitation of bacteria in blood and vegetations were generally the same as those described in detail elsewhere (Thompson et al. 1976). Briefly, endocardial vegetations were isolated aseptically, weighed, and homogenized in glucose broth, after which serial io-fold dilutions were made and ioo-il samples were plated on sheep blood agar and incubated for 24 or 48 h at 37TC. In the clearance experiments, 2-ml blood samples were taken from the central ear artery. These samples, prevented from clotting by dilution in i ml liquoid, were plated in 5 ml glucose agar broth and incubated for 24 or 48 h at 3 70C. Bacteraemia was expressed as the number of CFU per ml of blood. Since the detection limit for the blood cultures is i CFU per ml, for the statistical calculations sterile cultures were given a log1o value of - i.oo (i.e. i CFU per I0 ml). Serum. Serum for phagocytosis and intracellular killing experiments in vitro was pre-
pared from blood collected from the central ear artery of normal rabbits. The blood was allowed to clot for i h at room temperature and then centrifuged for I5 min at I500 g after which the supernatant serum was sterilized by filtration through a o.45-,um filter (Millipore Filter Corperation, Bedford, Mass.). The sera were pooled and stored in i-ml aliquots at - 20°C until use.
Polymorphonuclear leucocyte suspensions. For the in-vitro assessment of phagocytosis and intracellular killing, exudates were elicited in rabbits by an intraperitoneal injection of 75 ml sterile 5% glycogen solution (BDH). Fifteen hours later, the exudates were har-
259
vested with I50 ml sterile phosphate-buffered saline (PBS, pH 7.2) containing 50 iu heparin per ml. The average yield was 2 X io8 cells per rabbit. The cells were layered on Ficoll-Hypaque (density I.0 77) and centrifuged for 20 min at 420 g. The pellet containing granulocytes was washed twice and resuspended in Hanks' balanced salt solution (HBSS, pH 7.2) with o. I% (w/v) gelatin (Difco) (Gelatin-HBSS) to a final concentration of I. 5 X io7 cells per ml, of which on average 90% were granulocytes and io% mononuclear cells (macrophages and lymphocytes). Siliconized glass tubes were used throughout.
Phagocytosis assay. Phagocytosis assessment was done as described elsewhere in detail (van Furth et al. I978; Leijh et al. 1977). Briefly, 5 x io6 peritoneal granulocytes and 5 x io6 streptococci per ml in gelatine-HBSS (total volume 2 ml) were incubated in the presence of various concentrations of serum at 3 70C under slow rotation (4 r/min). At various time-points a o.5-ml sample of the suspension was added to I.5 ml ice-cold gelatin-HBSS to stop phagocytosis. After 4 min of centrifugation at I I0 g to spin down the granulocytes, the number of viable bacteria in the supernatant was determined by a
microbiological plate method. Phagocytosis is expressed as the percentage decrease of the initial number of viable bacteria, calculated according to the formula: Ft=
(i-Nt/No)xioo
in which Ft is the phagocytic index at t min, No the number of viable bacteria at o min, and Nt the number of viable extracellular bacteria at t min (van Furth et al. I978).
Intracellular killing assay. Intracellular killing of bacteria was determined independently of phagocytosis as described elsewhere (van Furth et al. I978; Leijh et al. I980). Briefly, streptococci pre-opsonized for 30 min in I0% serum, were phagocytosed for 3 min at a bacteria-to-cell ratio of I: I at 3 7°C under
260 M.J.M. Meddens et al. slow rotation in the absence of serum. The log1oyt = log1Oa-b log1ot, granulocytes containing ingested streptostarting at time point t = i min. In this cocci were separated from extracellular micequation Yt represents the number of CFU per ro-organisms by differential centrifugation at at time-point t min. Parameter a ml of blood I IO g for 4 min and two washes at 40C. Next, represents the number of CFU per ml of blood 5 x I06 granulocytes per ml (total volume 2 at time and b characterizes the rate of zero, ml) were re-incubated in i o% serum at 3 70C clearance. These parameters were used to under rotation. At various time-points the compare clearance in the various experinumbers of viable intracellular streptococci were determined after disruption of the mental groups. Three to five rabbits were granulocytes. The intracellular killing was used for each experimental group. Differences between the dextran-positive expressed as the percentage decrease of the and dextran-negative streptococci with reinitial number of viable intracellular streptoto phagocytosis and intracellular killspect cocci. This killing index was calculated ing, effect of the serum concentration, and according to the formula: growth properties, were estimated by an Kt= N/NO x ioo analysis of covariance (Nie et al. I 9 7 5). in which Kt is the intracellular killing index Results at t min, No the number of viable intracellular streptococci at o min, and Nt the number Effect of HN2 treatment on the numbers of of viable intracellular bacteria at t min. peripheral blood granulocytes and monocytes Clearance studies. The clearance from the To induce granulocytopenia HN2 was incirculation of the native Streptococcus sanguis jected intravenously (Fig. I). The number of and its dextran-negative mutant was mea- granulocytes per mm3 of blood decreased to sured after intravenous injection of i ml of a almost zero on day 3 when the steptococci suspension containing I05 CFU of either were injected. The granulocyte numbers strain into non-catheterized control and remained at this low level at least until the HN2-treated rabbits, in the last 3 or 4 days isolation of the vegetations 2 days later. The after an injection of HN2. Blood samples numbers of monocytes per mm3 of blood were drawn at I 5 s, and i, 3, 6, 9, I2 and 24 decreased to almost zero on day o, remained min thereafter. Numbers of CFU per ml were at this low level for 24 h, and then increased determined as described above. from day i to day 2. The numbers of granulocytes and monocytes per mm3 of Statistical analysis. The ID50 was calculated blood were significantly lower in the HN2according to Spearman & Kairber (Finney treated than in the control rabbits at the time I962). Comparison of two ID50s was per- of injection ofthe bacteria and all subsequent formed by dividing the difference between time-points. There were no significant differthem by the standard deviation of this differ- ences of the granulocyte or monocyte ence. The quotient has an approximately numbers between control or HN2-treated normal distribution from which P values rabbits injected with either dextran-positive were derived. or dextran-negative streptococci. The numbers of CFU per ml of blood determined in the clearance studies were Induction of endocarditis with dextran-positive analysed by fitting the data in the power or dextran-negative streptococci in control and curve: HN2-treated rabbits Yt = atrb To find out whether the production of dexby using the equation: tran by the Streptococcus sanguis could in-
26I Role of granulocytes in experimental endocarditis rIneeded to colonize the vegetations in 50% of I cathetter V t h the rabbits, which is a significant difference
L E E
5
(P< O.OI).
Lo 4 0. Q
'5U) 3 0 cn
S2
o1-
j m
E 5
E E
4
I;-
0,
0 1-
0 co
10
1
N
N
0
0 L.
.
-4
-3
0
1
2
Days Fig. i. Effect of HN2 on the niumbers of blood granulocytes and monocytes durwing the course of dextran-positive and dextran-negative Streptococcus sanguis endocarditis. The vertical bars represent the SEM; the numbers at time-point day -4 represent the number of rabbits per treatment group. Rabbits were inoculated with I03-IO5 CFU. On days o, i, and 2, the numbers of granulocytes or monocytes were significantly lower in the HN2-treated than in the control rabbits (Po.so), but was significantly lower for the dextran-positive than the dextran-negative streptococci (P < o. ooI ). From I to 5% serum, the phagocytic indexes for dextran-positive streptococci were still significantly lower (Fig. 2, P< 0.00s), but in this range increasing serum concentrations led to an increased phagocytic index for both strains (P 0.4). At higher serum concentrations no further increase of the phagocytic index was found (data not shown).
Growth properties of the dextran-positive and dextran-negative streptococci without phagocytic cells in vitro To find out whether differences in the extracellular growth rate of both strains could contribute to the differences in phagocytosis observed with various serum concentrations, dextran-positive and dextran-negative streptococci were incubated at 3 70C under slow rotation in the presence of o and io% rabbit serum for 120 min. No differences were found between the growth rates of the two strains in either
Role of granulocytes in experimental endocarditis
263
Table 3. Clearance of dextran-positive and dextran-negative Streptococcus sanguis in control and HN2-treated rabbits
Clearance parameters Analysis
Experimental group log1oa
A
control: D (+)74 HN2:
B
D(-) D(+)
3.40
Difference between D (+) and D(-) -b t (P-value) 4.20
2.40 I.69
3.33
D(-)
2.40
r2 0.82
(P=O.OOOI) 4.04 (P=0.0002)
o.8o
Rabbits were inoculated with IO5 CFU of one of the strains. Three to five rabbits were used in each group. Between control and HN2-treated rabbits logloa and b did not differ significantly (P> 0.5o). D (+), dextran-positive Streptococcus sanguis: D (-), dextran-negative mutant.
Table 4. In-vitro phagocytosis and intracellular killing of dextran-positive and dextran-negative Streptococcus sanguis by rabbit peritoneal granulocytes
100 of% ov -s
x
Killing indext
Phagocytic
indext
Time* D (+) D(-) D(+) 0
30 60 I20
0
0
48.7 42.5 76.7 75.5 89.I 89.o
100
31.3 34.9
1I7.0
0
60' W40 -,..
D(-) I00
48.5
I5.7 6.5
* Minutes after the start of the phagocytosis or killing experiments. t Expressed as indicated under materials and methods. Values are mean of three experiments. Analysis of covariance showed no difference between the two strains for the phagocytosis (P < 0.40) or the intracellular killing (P>o.5o).
XL
20
0 6 4 8 10 2 Serum concentration (%) Fig. 2. Effect of serum concentration on the ability of peritoneal exudate granulocytes to phagocytose dextran-positive (0) and dextran-negative (0) Streptococcus sanguis. Values at each point represent the mean of three experiments; the phagocytic index F60 determined at 60 min after the start of phagocytosis is explained under materials and methods. Bacteria-to-cell ratio = 0
264
M.J.M. Meddens et al. HBSS or in HBSS supplemented with i o% these animals there was no difference in ID50 serum (P>o.65). between the two strains. This makes it less likely that the difference in clearance could the different ID50s in the control explain Ability of granulocytes to kill dextran-positive rabbits. and dextran-negative streptococci intracelluAnother explanation for the different ID5Os larly could be that dextran hampers the phagocyTo find out whether the production of dex- tic removal of streptococci from the vegetational surface by granulocytes. This could be tran could influence the intracellular killing of streptococci, the ability of granulocytes to due to a tighter adherence of the streptococci kill dextran-positive and dextran-negative to the vegetations. Alternatively, phagocytosis could be impaired by dextran, which streptococci was studied in vitro. The results of the experiments show that would retard the elimination of dextranafter phagocytosis the granulocytes killed bearing streptococci. The latter possibility the dextran-positive and the dextran-nega- might be supported by the in-vitro finding that at serum concentrations below i o% and tive streptococci equally well (Table 4). in the absence of serum granulocytes phagocytosed less of the dextran-positive streptoDiscussion coccus than of the dextran-negative mutant, In the present study the effect of HN2 although at serum concentrations of i o% and higher there was no difference between treatment on the induction of endocarditis by a dextran-producing Streptococcus sanguis the phagocytosis of the two strains. This and its dextran-negative mutant was investi- raises the question whether the differences gated. HN2 treatment leads to both granulo- observed in vitro can explain the differences in ID50 in vivo where bacteria are opsonized cytopenia and monocytopenia. However, since monocytes are known to have no effect in undiluted serum. However, it probably on either the induction or course of Strepto- only takes a few minutes for intravenously injected streptococci to become attached to coccus sanguis endocarditis (Thorig et al. I980), the effect of HN2 treatment can be the vegetations and covered by fibrin, and it attributed to its granulocyte-depleting acti- is only during this very short period that the vity. phagocytes can remove the streptococci from In the control rabbits the ID50 was signifi- the vegetational surface. During this interval cantly lower for the dextran-positive strepto- opsonization might be incomplete, and thus coccus than for the dextran-negative the conditions for phagocytosis would be mutant, but in the granulocytopenic rabbits similar to those under low serum concentrathere was no difference for the ID50 between tions in vitro. The clearance from the circulathe strains. However, for both strains the tion of the dextran-positive steptococci was ID50 was significantly lower in the granulo- found to be significantly slower than that of cytopenic than in the control rabbits which the dextran-negative mutant. These microsuggests that granulocytes play a role during organisms are most probably cleared by cells the induction of endocarditis. The different of the mononuclear phagocyte system and ID50s in the control rabbits might be the mononuclear phagocyte system and explained by a dextran-dependent difference therefore, the observed difference of the in clearance. Indeed, we found that the clearance could be an additional argument clearance of the dextran-positive strepto- in favour of an effect of dextran on phagococcus was significantly lower than that of cytosis in vivo. the dextran-negative mutant in the control The ID50 was significantly higher in conrabbits. However, the same difference was trol rabbits than in HN2-treated rabbits, found in the granulocytopenic rabbits, but in which was not found in a previous study
Role of granulocytes in experimental endocarditis 265 LEIJH P.C.J., VAN (Meddens et al. I 982; Th6rig et al. I 980). A DEN BARSELAAR M.TH., VAN ZWET TH.L. & VAN FURTH R. (I 9 7 7) Kinetics of simple explanation for this discrepancy canphagocytosis of Staphylococcus aureus and not be offered, but differences in experimenEscherichia coli by human granulocytes. Imtal conditions could have contributed, such munology 37, 453-465. as the strain of rabbits used or a possibly LEIJH P.C.J., VAN DEN BARSELAAR M.TH., DUBBELDEdifferent surface condition of the injected MAN-REMPT I. & VAN FURTH R. (I980) Kinetics streptococci due to the relatively rich nutrient medium used in the present investi-
gation. In sum, this study provides arguments that a dextran-dependent defective removal of bacteria from the vegetational surface due to either an impaired phagocytosis or a tighter adherence of bacteria can be a factor promoting the induction of endocarditis by dextran-producing streptococci.
Acknowledgements The authors are greatly indebted to Mr N. Soedirman of the National Institute of Public Health (Bilthoven, The Netherlands) for the selection of the dextran-negative mutant streptococcus. The skilful technical assistance of Mrs W.C. Schmall-Bauer and Mrs T.L. van Zwet is gratefully acknowledged. This investigation was supported by grant 78IOI from the Dutch Heart Association (The Hague). References ANGRIST A.A. & OKA, M. (I963) Pathogenesis of bacterial endocarditis. J. Am. med. Ass. I83, 249.
DURACK D.T. & BEESON P.P. (I972) Experimental bacterial endocarditis. I. Colonization of a sterile vegetation. Br. J. exp. Path. 53, 33-49. FINNEY D.J. (I962) Probit Analysis. Cambridge: University Press. GOULD K., RAMIREz-RONDA C.H., HOLMES R.K. & SANFORD J.P. (I975) Adherence of bacteria to heart valves in vitro. 1. clin. Invest. 56, 1364-1370. GUTHOFF 0. (1970) Vorkommen und Nachweiss von Ektopolysaccharide bei Streptokokken. Zentbl. Bakt. I 21I5, 43 5. HEHRE E.J. & NEIL J.M. (1946) Formation of serological reactive dextrans by streptococci from subacute bacterial endocarditis. 1. exp. Med. 83, 147.
of intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes. Eur. J. Immun. 10, 750-757. MEDDENS M.J.M., THOMPSON J., EULDERINK F., BAUER W.C., MATrIE H. & VAN FURTH R. (I982) Role of granulocytes in experimental Streptococcus sanguis endocarditis. Infect. Immun. 36, 325-332. NIE N.H., HADLAY HULL C., JENKINS J.G., STEINBRENNER K. & BENT D.H. (I975) Statistical Package for the Social Sciences. 2nd Edn, release 7-9. New York: McGraw Hill Book Company. PELLETIER L.L., COYLE M. & PETERSDORF R. (I978) Dextran production as a possible virulence factor in streptococcal endocarditis. Proc. Soc. exp. Biol. Med. 158, 415-420. RAMIREZ-RONDA C.H. (I978) Adherence of glucan-positive and glucan-negative streptococcal strains to normal and damaged heart valves. l. clin. Invest. 62, 805-8I4. SCHELD W.M., VALONE J. & SANDE M.E. (1978) Bacterial adherence in pathogenesis of endocarditis. J. clin. Invest. 6i, 1394-1404. STATISTIcS APPLICATIONS (1978) HP-33E. Singapore: Hewlett-Packard Company. THOMPSON J., EULDERINK F., LEMKES H. & VAN FURTH R. (1976) Effect of warfarin on the
induction and course of experimental endocarditis. Infect. Immun. 14, 1204-1209 THORIG L., THOMPSON J., EMEIS 1.J. & VAN FURTH R. (I980) Effects of monocytopenia and anticoagulation in experimental Streptococcus sanguis endocarditis. Br. 1. exp. path. 6i, io8-i i6. VAN FURTH R., VAN ZWET T.L. & LEIJH P.C.J. (I 9 78) In vitro determination of phagocytosis and intracellular killing by polymorphonuclear phagocytes. In Handbook of experimental Immunology. 3rd Edn, Ed. D.M. Weir. Oxford: Blackwell Scientific Publications, Ch. 32, pp. 1-19. VAN GINKEL C.J.W., THbRIG L., THOMPSON J., OH J.I.H. & VAN AKEN W.G. (I 9 79) Enhancement of generation of monocyte tissue thromboplastin by bacterial phagocytosis: possible pathway for fibrin formation on infected vegetations in
bacterial endocarditis. Infect. Immun. 2 5, 388-395.
