- Email: [email protected]
Platelet aggregation induced by uncommon serotypes of Streptococcus sanguis isolated from patients with Behçet's disease
0003-9969/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc
Arch oral Bid. Vol. 36, No. 6, pp. 425-429, 1991 Printed in Great Britain. All rights reserved
PLATELET AGGREGATION INDUCED BY UNCOMMON SEROTYPES OF STREPTOCOCCUS SANGUIS ISOLATED FROM PATIENTS WITH BEHCET’S DISEASE EMIKO ISOGAI,~HIROSHIISCKZAI~KEIJI YOKOTA,~SHUNJI HAYASHI,~ NOBUHIRO FUJII,~ KELJIOGUMA,’ KOJI YOSHIRAWA,~ YOICHI SASAMOTO, SATOSHIKOTAICE~and SHIGEAKIOHNO~
‘Department of Preventive Dentistry, School of Dentistry, Higashi Nippon Gakuen University, IshikariTobetsu 1757, Hokkaido 061-02, *Division of Animal Experimentation and 3Department of Microbiology, Sapparo Medical College, Sapporo 060, 4Department of Ophthalmology, Hokkaido University School of Medicine, Sapporo 060 and Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama 232, Japan (Accepied 16 January 1991) Summary-Uncommon serotypes were tested for their ability to induce aggregation in platelet-rich plasma. Both uncommon and common serotypes induced platelet aggregation in rabbit platelet-rich plasma, but serotonin release was higher with the uncommon serotypes. Aggregation at ATP release varied between uncommon and common serotypes. With human platelet-rich plasma, only uncommon serotypes showed aggregation. Differences in serotype selectivity and the rate of aggregation were noted among platelet donors. About half of the patients with Behcet’s disease and 30% of health controls showed platelet aggregation. Plasma from non-responder patients with Behcet’s disease inhibited aggregation of healthy responder platelets within 20 min. Thus selective binding of uncommon serotypes of Strep. sunguis to plateletes might cause the vasculitis in Behqet’s disease. Key words: platelet, uncommon serotypes of Strepfococcus sanguis, Behqet’s disease..
INTRODIJffION
The aetiology of Behcet’s disease has not been established. Attempts have been made to identify micro-organisms in patients with this disease (Barile et al., 1963; Graykowski et al., 1966; Yamanouchi et al., 1981; Eglin, Lehner and Subak-Sharpe, 1982). Recently, we found that (1) the oral flora of patients with Behcet’s disease was different from that of controls, (2) the proportion of Strepcoccus sanguis in the flora of patients with Beh9et’s disease was significantly higher than that of controls and (3) that the serotypes of isolated Strep. sanguis were different from those of the typical Groups I-IV (common serotypes), (Isogai et al., 1990a, b; The Beh9et’s Disease Research Committee of Japan, 1989). Furthermore, (4) the serum antibody titre and intensity of skin reaction against these organisms were higher in Behcet’s disease than in controls, and (5) the proportion of Strep. sanguis in the oral flora showed significant correlation with the level of chemiluminescent response of neutrophils. Interaction between platelets and certain bacteria are likely to be part of the pathogenesis of diseases such as bacterial endocarditis (Herzberg et al., 1990; Herzberg, MacFarlane and Delzer, 1985). Strep. sanguis serotypes interact selectively with human platelets. Interactive strains of Strep. sunguis were frequently isolated from patients with endocarditis (Herzberg, Brinzenhofe and Clawson, 1983). It has been suggested that there may be receptors for certain stains of Strep. sanguis on human platelets (Herzberg et ai., 1985).
The pathogenesis of subacute bacterial endocarditis has been studied in animal models. The viridans groups of streptococci adhere selectively to preformed sterile vegetations in viva (Freedman and Valone, 1979; Hook and Sande, 1974) and to platelet-fibrin clots in vitro (Scheld, Valone and Sande, 1978). It has been suggested that microorganisms that have surface dextrans adhere better and are more virulent (Ramirez-Ronda, 1978, 1980; Sheld et al., 1978). It is important to determine whether the uncommon serotypes of Strep. sanguis activate and aggregate platelets. We have now examined aggregation and release of ATP in platelet-rich plasma from rabbits and humans by uncommon serotypes isolated from patients with Beh@‘s disease. Serotonin release from rabbit platelets was also measured. MATERIALS AND METHODS
Bacterial strains Strep. sanguis strain 113-20 (uncommon serotype I), 114-23 (uncommon serotype III) and 118-l (uncommon serotype IV) were used after isolation from contirmed cases of Behpt’s disease. Strep. sanguis SSH-83 (uncommon serotype I) and KIH-T (uncommon serotype II) were from patients with Kawasaki disease. Strep. sunguis ATCC 10556 (common serotype I), ATCC 10557 (common serotype II), ATCC 10558 (common serotype III) and ST-7 (common serotype IV) were also used.
425
426 Growth conditions suspensions
EMIKO
and preparation
of
IWGAI
bacterial
Bacteria were harvested by centrifugation from late stationary-phase growth (18 h) in Todd-Hewitt broth (59OOg for 20 min at 4°C). Cells were washed four times in cold Hanks balanced salt solution (Nissui Pharamaceutical Co. Ltd, Japan). After washing, the bacteria were dispersed by vortex mixing for 20 s and suspended to an absorbance at 620nm (&,,) of approx. 2.0 (containing 4 x lo9 cells/ml), as determined by counting in Petroff-Hausser chamber. Preparation of platelets
Rabbit blood was collected in 0.1 vol of 129 mmol/l sodium citrate. Citrated blood was gently mixed, then centrifuged at 150g for 10 min at room temperature. Platelet-rich plasma was removed and the platelet concentration was adjusted to 2.5 x lo9 by addition of platelet-poor plasma obtained by centrifuging the platelet-rich plasma at 1800 g for 10 min. The platelet number was counted and the platelet-rich plasma was stored in capped plastic tubes at room temperature; the experiments were carried out within 4 h. Human platelet-rich and -poor plasma was prepared in same way.
et
al.
count after lysis with Triton X-100. Backgrounds, which normally were 410% of the total, were subtracted from the experimental results. The data are presented as the mean of triplicate samples. Platelet aggregation and release of ATP
A lumi-aggregometer (AHS/Japan Co.) was used to measure both rabbit and human platelet aggregation and release of ATP. In each experiment, plateletpoor plasma was used to adjust the intrument for 100% transmission in order to represent 100% aggregation. A mixture of 450 ~1 of platelet-rich plasma, 50~1 of luciferin luciferase reagent (40 mg/ml in distilled water, Sigma Co., St Louis, MO, U.S.A.) and 5011 of HEPES-Hanks buffer was used to represent 0% transmission (zero aggregation). In the experiments, a mixture of 450 ~1 platelet-rich plasma and 50 ~1 luciferin luciferase reagent was added to a cuvette, which was placed in the lumi-aggregometer, stirred at lOOO/rev/min, and allowed to warm to 37°C. The bacterial solution was then added (50~1) and platelet aggregation and ATP release were recorded. A collagen standard (2 mg/ml, final concentration 2OOpg/ml) was used as a positive control. RESULTS
Release of serotonin
Release of serotonin was tested in rabbit platelets. Serotonin within platelets was labelled by incubation of 0.6 pCi/ml of [3H]-serotonin (S-hydroxy[G-3H]-tryptamine creatinine sulphate, l&20 Ci/mol; Amersham, Arlington Heights, IL, U.S.A.) for 20 min, at 37°C sedimented at 18OOg for 15 min, washed successively with HEPES-Hanks buffer and finally resuspended in the buffer at a concentration of 2.5 x lo9 platelets per ml. Assays of serotonin release was routinely performed using 100 ~1 of platelet solution, 250 ~1 of plasma, 50 ~1 of HEPES-Hanks buffer, and 100 ~1 of the bacterial solution. Reactions were carried out in polystyrene tubes at 37°C for 30, 60 and 90 min, respectively. The tubes were then cooled to 4°C and centrifuged at 18OOg for 15 min. Supernatants were assessed for [3H]-serotonin and were expressed as a percentage of the total released
Rabbit platelet aggregation, release of ATP, and serotonin release induced by Strep. sanguis
and common serotypes of Strep. rabbit platelet aggregation and release of ATP as effectively as standard collagen. However, there were quantitative differences in the reactions elicited by these streptococci (Table 1). Strain 118-1, which was isolated from a patient with Behcet’s disease, induced the greatest aggregation/ ATP release ratio, and the shortest lag phase. Other isolates from patients with the disease (113-20 and 114-23) showed similar activities to those of the common serotypes. Serotonin release, after rabbit platelets were exposed to various serotypes of Strep. sanguis, are shown in Fig. 1. Uncommon serotypes induced greater release of serotonin than common serotypes; Both uncommon
sanguis induced
Table 1. Rabbit platelet aggregation and release of ATP by Strep. sanmis Strep. smguis Serotype
Strain
uncommon I 113-20 SSH-83 II KIH-T III 114-23 IV 118-l Common I ATCC 10556 II ATCC 10557 III ATCC 10558 IV ST-7 Collagen
Aggregation (%)
ATP release ratio”
Lag time (min)
54.3 * 17.0b 28.7 + 14.5* 21.3 + I3.5* 71.Ok22.1 93.7* 2.1**
0.82 + 0.37 0.35 & 0.17 0.30 & 0.26 3.64 f 2.23 4.505 1.44
2.0 * 0.5 3.3 * 0.3 2.4* 1.2 1.5*05 0.7 + 0.1
70.3 + 19.3 22.3 k 14.1* 53.3 f 11.7 67.0 + 11.4 60.3 &-11.2
0.17 f 0.08 0.56 f 0.15 0.94 10.45 3.06 + 1.59 1.00
1.9 * 1.2 2.2 _+0.3 2.1 f 0.6 1.5 + 0.5 1.0*0.5
aRatio of Strep. sanguis stimulation/collagen stimulation. bResults are given as mean (+ SD). *Significantly lower than % aggregation with collagen, p < 0.01. **Significantly higher than % aggregation with collagen, p < 0.01.
Platelet activation by Strep. sun@
427
human platelet-rich plasma. Strep. sanguis strains originally isolated from patients with Behqet’s or Kawasaki disease, unlike the common strains, promote platelet aggregation (Table 2). However, differences in the aggregation of platelet-rich plasma were observed among human donors. Responder frequency was 50% in patients with Behyet’s disease and 30% in controls. The levels of platelet aggregation at ATP release in responders are shown in Table 3. Aggregation in patients with Behyet’s disease was lower than that in healthy controls. However, aggregation induced by standard collagen was significantly higher in the patients (Table 4). Similar results were found for ATP release. Eflect of plasma from patients with Behget’s disease
0
30
Time
after
incubation
Plasma from non-responders with Behqet’s disease inhibited both aggregation and ATP release. The
90
60
15
(min)
Fig. 1. Serotonin release from rabbit platelets after stimulation with Sfrep. sanguk. 0: uncommon serotype I, upper, SSH-83, lower, 113-20; v: uncommon serotype II, KIH-T; A: uncommon serotype III, 114-23; m: uncommon serotype IV, 118-l; 0: common serotype I, ATCC 10556; V: common serotype II, ATCC 10557; A: common serotype. III, ATCC 10558; 0: common serotype IV, ST-7. a rapid increase
was found within
15 min, and after
30min the responses reached plateau. Human platelet aggregation and release at ATP induced by uncommon serotypes of Strep. sanguis
Strains of Strep. sanguis were tested for their abilities to promote platelet aggregation in fresh
inhibition was complete within 20min but over a longer period of observation, the platelet response could reappear. The levels of aggregation and ATP release were about half of those of the original levels. DISCUSSION
We show that only uncommon serotypes of Strep. platelets. Herzberg et al. (1985) have reported that Strep. sanguis is selectively recognized and bound by platelets, which respond physiologically. Surface component class I of Strep. sanguis mediates direct binding to a platelet-binding site (step 1). After adherence, the class II component of Strep. sanguis mediates Ca*+-dependent platelet
sanguis aggregate human
Table 2. Responsiveness of human platelets to Sfrep. sanguis
Platelets from
Subjects reacting with
Strep. smguis (%)
Responder ratio
113-20
SSH-83
114-23
118-l
Others”
s/10
40
40
10
20
0
10
30
10
10
0
Patients with Reh9et’s disease
NSb Healthy controls
3/10
aCommon serotypes I (ATCC 10556), II (ATCC 10557), III (ATCC 10558) and IV (ST-7). bNS: not significant. Table 3. Platelet aggregation and ATP release level in responders Strep. sanguis Platelets from
113-20
Patients with Behcet’s disease
Healthy controls
Aggregation (%) 16.0 k 13.4 Lag time (mm) 19.8 + 7.6 ATP release 0.13 f 0.10 ratio Aggregation (%) 40 Lag time (min) 11.5 ATP release
SSH-83
114-23
118-I
11.3 + 3.0 18.0 + 6.0 0.12 * 0.07
8 13
9.5 21
51.3 i24.8 11.3 * 1.2 0.19
14 17 -
22 15.8 -
Results are given as mean (k SD). Table 4. Platelet aggregation induced by collagen Platelets from Patients with Behcet’s disease Healthy controls *p <
Aggregation (%) ATP release level 83.0 f 6.3’ 65.9 f 13.9*
199.1 f 253.6* 65.9 f 13.9*
0.01; NS: not significant, unpaired two samples r-test.
Lag time (min) 0.97 & 0.34 NS 1.07 f 0.39 NS
428
EMIKOISOGAI
activation (step 2). The class III component may be an exo-ATPase that hydrolyses ATP (step 3). In our experiments, only clinical isolates (uncommon serotypes) activated human platelets, implying that there are distinct receptors for certain Strep. sang& strains on these cells. Selective platelet aggregation may contribute to the vasculitis occuring in Behget’s disease. Platelets contain a number of organelles that store polypeptides and low molecular-weight components. These organelles release their components after activating the clotting process. The a-granules containing plateletderived growth factor, @-thromboglobulin and fibrinogen. The primary in uiuo function of plateletderived growth factor is to induce mitosis in quiescent cells such as diploid fibroblasts and arterial smoothmuscle cells. This would corroborate the proposed in vivo role of this growth factor in wound healing. The ability of the factor to stimulate proliferation and migration of arterial smooth-muscle cells, and to stimulate synthesis of cholesterol esters prompted the suggestion that it might play a part in the pathogenesis of atherosclerosis (Ross and Glomset, 1976; Ross, 1981). In patients with Behvt’s disease, platelets that aggregated at the site of vascular injury may release the growth factor, which then initiates events involving ceI1 migrations and proliferation into the intima of arterial smooth-muscle cells. The process may lead to the formation of vascular lesions. About 30% of patients with Behget’s disease have vasculitis, and this may be related to platelet activation. We found that rabbit platelets were aggregated by all strains of Strep. sang&, although clinical isolates caused a greater release of serotonin than did the common strains. The differences in the results between serotonin release and aggregation/ATP release, may indicate methodological differences. The indirect method such as radiolabelling may be relevant only if non-thrombogenic surfaces are used and/or if platelet aggregation is inhibited (Sakariassen, Bolhuis and Sixma, 1979). From our experiments, we suggest that the origin of platelets and their bioassay are of importance in the investigation of the role of platelet abnormalities in Behvt’s disease. The coagulation system may augment immunologically mediated tissue injury in infections and autoimmune diseases (Naish, Penn and Evans, 1972; Vassalli and McCluskey, 1971). We found variation in both selectivity and rate of response among the platelet-rich plasmas from donors. Plasma from nonresponders delayed the platelet response of responders, indicating that plasma factors may influence the response. Such factors may be present in those patients with BehGet’s disease in whom the platelet response to the uncommon serotypes of Strep. sanguis was depressed, although the collagen-stimulated platelet response was significantly higher than in controls. Thus, the induction of Behcet’s disease may require infection with particular strains, or a mixture of Strep. sanguis, as well as the presence of appropriate receptors in platelets. In addition, plasma factors may effect the pathogenesis of this disease. Acknowledgemenr-These studies were supported by the Research Committee of Behqet’sdisease in Japan,
et al. REFERENCES
Barile M. F., Graykowsky E. A., Driscoll E. J. and
Rigges D. B. (1963) L form of bacteria isolated from recurrent aphthous stomatitis lesions. Oral Surg. 16, 139551402. Eglin R. P., Lehner T. and Subak-Sharpe J. H. (1982) Detection of RNA complementary to Herpes-simplex virus in mononuclear cells from patients with Behpt’s syndrome and recurrent oral ulcers. Luncet II, 1356-1360. Freedman L. R. and Valone J. Jr (1979) Experimental infective endocarditis. Prog. car&wax. Dis. 22, 169%180. Graykowski E. A., Barile M. F., Lee W. B. and Stanley H. R. (1966) Recurrent aphthous stomatitis. J. Am. med. Ass. 1%. 637644.
Herzberg M., C., Brinzenhofe K. L. and Clawson C. C. (1983) Aggregation of human platelets and adhesion of Streptococcus sanguis. Infect. Immun. 39, 1457-1469.
Herzberg M. C., Gong K., MacFarlane G. D., Ericson P. A., Soberay A. H., Krebsbach P. H., Manjula G., Schilling K. and Bowen W. H. (1990) Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis. Infect. Immun. 58, 512-522. Herzberg M. C., MacFarlane G. D. and Delzer P. R. (1985) Streptococcus sanguis interaction with human platelets. In Molecular Basis of Oral Microbial Adhesion (Eds Mergenhagen S. E. and Rosan B.)., RR. 53-60. American Society fo; Microbiology, U.S.A. __ Hook E. W. III and Sande M. A. (19741 Role of the vegetation in experimental Streptocdccus &idans endocarditis. fnfecf. Immun. 10, 1433-1438. Isogai E., Ohno S., Kotake S., Isogai H., Tsurumizu T., Fujii N., Yokota K., Syuto B., Yamaguchi M., Matsuda H. and Oguma K. (1990a) Chemiluminescence of neutrophils from patients with Behqet’s disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora. Archs oral Biol. 35, 4348.
Isogai E., Ohno S., Takeshi K., Yoshikawa Y., Tsurumizu T., Isogai H., Yokata K., Kotake S., Sasamoto Y., Hashimoto T., Shimizu H., Matsuda H., Fujii N., Yamaguchi M. and Oguma K. (1990b) Close association of Streptococcus sanguis uncommon serotypes with Behqet’s disease. BiJidobact. Microjora. 9, 27-24. Mayrand D. (1985)Virulence promotion by mixed bacterial infections. In The Pathonenesis of Bacterial Infections (Eds Jackson G. G. ad Thomas H.), pp. i81-291.
Springer, Berlin. Naish P., Penn G. B. and Evans D. J. (1972) The effect of difibrination on nephrotoxic serum nephritis in rabbits. Clin. Sci. 42, 643-646.
Ramirez-Ronda C. H. (1978) Adherence of glucan-positive and glucan negative streptococcal strains to normal and damaged heart valves. J. clin. Invest. 62, 805-814. Ramirez-Ronda C. H. (1980) Effect of molecular weight of dextran on the adherence of Streptococcus sanguis to damaged heart valves. Infect. Immun. 29, 1-7. Ross R. (1981) Atherosclerosis: a problem of the biology of arterial wall cells and their interaction with blood components. Atherosclerosis 1, 293-3 Il. Ross R. and Glomset N. (1976) The pathogenesis of atherosclerosis. New Engl. J. Med. 295. 369-376. Sakariassen K. S., Bolh& P. A., and Sixma J. J. (1979) Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium. Nature (Lond.) 279, 636638.
Scheld W. M., Valone J. A. and Sande M. A. (1978) Bacterial adherence in the pathogenesis of endocaiditis. J. din. Invest. 61, 1394-1404.
Platelet activation by Srrep. swguis The Beh@‘s Disease Research Committee of Japan (1989) Skin hypersensitivity to streptococcal antigens and the induction of systemic symptoms by the antigens in Behcet’s disease-a multicenter study. J. Reumatol. 16, 506-511. Vassalli P. and McCluskey R. T. (1971) The pathogenie role of the coagulation process in glomerular
429
disease of immunological origin. Adu. Nephrol. 1, 41-63. Yamanouch K., Shishido A., Kobune F., Sakota H. and Inaba G. (1981) Virological approaches to Behcet’s disease in Japan. In Behget’s Disease (Ed. Inaba G.), pp. 57-63. Univ. of Tokyo Press, Tokyo.
Arch oral Bid. Vol. 36, No. 6, pp. 425-429, 1991 Printed in Great Britain. All rights reserved
PLATELET AGGREGATION INDUCED BY UNCOMMON SEROTYPES OF STREPTOCOCCUS SANGUIS ISOLATED FROM PATIENTS WITH BEHCET’S DISEASE EMIKO ISOGAI,~HIROSHIISCKZAI~KEIJI YOKOTA,~SHUNJI HAYASHI,~ NOBUHIRO FUJII,~ KELJIOGUMA,’ KOJI YOSHIRAWA,~ YOICHI SASAMOTO, SATOSHIKOTAICE~and SHIGEAKIOHNO~
‘Department of Preventive Dentistry, School of Dentistry, Higashi Nippon Gakuen University, IshikariTobetsu 1757, Hokkaido 061-02, *Division of Animal Experimentation and 3Department of Microbiology, Sapparo Medical College, Sapporo 060, 4Department of Ophthalmology, Hokkaido University School of Medicine, Sapporo 060 and Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama 232, Japan (Accepied 16 January 1991) Summary-Uncommon serotypes were tested for their ability to induce aggregation in platelet-rich plasma. Both uncommon and common serotypes induced platelet aggregation in rabbit platelet-rich plasma, but serotonin release was higher with the uncommon serotypes. Aggregation at ATP release varied between uncommon and common serotypes. With human platelet-rich plasma, only uncommon serotypes showed aggregation. Differences in serotype selectivity and the rate of aggregation were noted among platelet donors. About half of the patients with Behcet’s disease and 30% of health controls showed platelet aggregation. Plasma from non-responder patients with Behcet’s disease inhibited aggregation of healthy responder platelets within 20 min. Thus selective binding of uncommon serotypes of Strep. sunguis to plateletes might cause the vasculitis in Behqet’s disease. Key words: platelet, uncommon serotypes of Strepfococcus sanguis, Behqet’s disease..
INTRODIJffION
The aetiology of Behcet’s disease has not been established. Attempts have been made to identify micro-organisms in patients with this disease (Barile et al., 1963; Graykowski et al., 1966; Yamanouchi et al., 1981; Eglin, Lehner and Subak-Sharpe, 1982). Recently, we found that (1) the oral flora of patients with Behcet’s disease was different from that of controls, (2) the proportion of Strepcoccus sanguis in the flora of patients with Beh9et’s disease was significantly higher than that of controls and (3) that the serotypes of isolated Strep. sanguis were different from those of the typical Groups I-IV (common serotypes), (Isogai et al., 1990a, b; The Beh9et’s Disease Research Committee of Japan, 1989). Furthermore, (4) the serum antibody titre and intensity of skin reaction against these organisms were higher in Behcet’s disease than in controls, and (5) the proportion of Strep. sanguis in the oral flora showed significant correlation with the level of chemiluminescent response of neutrophils. Interaction between platelets and certain bacteria are likely to be part of the pathogenesis of diseases such as bacterial endocarditis (Herzberg et al., 1990; Herzberg, MacFarlane and Delzer, 1985). Strep. sanguis serotypes interact selectively with human platelets. Interactive strains of Strep. sunguis were frequently isolated from patients with endocarditis (Herzberg, Brinzenhofe and Clawson, 1983). It has been suggested that there may be receptors for certain stains of Strep. sanguis on human platelets (Herzberg et ai., 1985).
The pathogenesis of subacute bacterial endocarditis has been studied in animal models. The viridans groups of streptococci adhere selectively to preformed sterile vegetations in viva (Freedman and Valone, 1979; Hook and Sande, 1974) and to platelet-fibrin clots in vitro (Scheld, Valone and Sande, 1978). It has been suggested that microorganisms that have surface dextrans adhere better and are more virulent (Ramirez-Ronda, 1978, 1980; Sheld et al., 1978). It is important to determine whether the uncommon serotypes of Strep. sanguis activate and aggregate platelets. We have now examined aggregation and release of ATP in platelet-rich plasma from rabbits and humans by uncommon serotypes isolated from patients with Beh@‘s disease. Serotonin release from rabbit platelets was also measured. MATERIALS AND METHODS
Bacterial strains Strep. sanguis strain 113-20 (uncommon serotype I), 114-23 (uncommon serotype III) and 118-l (uncommon serotype IV) were used after isolation from contirmed cases of Behpt’s disease. Strep. sanguis SSH-83 (uncommon serotype I) and KIH-T (uncommon serotype II) were from patients with Kawasaki disease. Strep. sunguis ATCC 10556 (common serotype I), ATCC 10557 (common serotype II), ATCC 10558 (common serotype III) and ST-7 (common serotype IV) were also used.
425
426 Growth conditions suspensions
EMIKO
and preparation
of
IWGAI
bacterial
Bacteria were harvested by centrifugation from late stationary-phase growth (18 h) in Todd-Hewitt broth (59OOg for 20 min at 4°C). Cells were washed four times in cold Hanks balanced salt solution (Nissui Pharamaceutical Co. Ltd, Japan). After washing, the bacteria were dispersed by vortex mixing for 20 s and suspended to an absorbance at 620nm (&,,) of approx. 2.0 (containing 4 x lo9 cells/ml), as determined by counting in Petroff-Hausser chamber. Preparation of platelets
Rabbit blood was collected in 0.1 vol of 129 mmol/l sodium citrate. Citrated blood was gently mixed, then centrifuged at 150g for 10 min at room temperature. Platelet-rich plasma was removed and the platelet concentration was adjusted to 2.5 x lo9 by addition of platelet-poor plasma obtained by centrifuging the platelet-rich plasma at 1800 g for 10 min. The platelet number was counted and the platelet-rich plasma was stored in capped plastic tubes at room temperature; the experiments were carried out within 4 h. Human platelet-rich and -poor plasma was prepared in same way.
et
al.
count after lysis with Triton X-100. Backgrounds, which normally were 410% of the total, were subtracted from the experimental results. The data are presented as the mean of triplicate samples. Platelet aggregation and release of ATP
A lumi-aggregometer (AHS/Japan Co.) was used to measure both rabbit and human platelet aggregation and release of ATP. In each experiment, plateletpoor plasma was used to adjust the intrument for 100% transmission in order to represent 100% aggregation. A mixture of 450 ~1 of platelet-rich plasma, 50~1 of luciferin luciferase reagent (40 mg/ml in distilled water, Sigma Co., St Louis, MO, U.S.A.) and 5011 of HEPES-Hanks buffer was used to represent 0% transmission (zero aggregation). In the experiments, a mixture of 450 ~1 platelet-rich plasma and 50 ~1 luciferin luciferase reagent was added to a cuvette, which was placed in the lumi-aggregometer, stirred at lOOO/rev/min, and allowed to warm to 37°C. The bacterial solution was then added (50~1) and platelet aggregation and ATP release were recorded. A collagen standard (2 mg/ml, final concentration 2OOpg/ml) was used as a positive control. RESULTS
Release of serotonin
Release of serotonin was tested in rabbit platelets. Serotonin within platelets was labelled by incubation of 0.6 pCi/ml of [3H]-serotonin (S-hydroxy[G-3H]-tryptamine creatinine sulphate, l&20 Ci/mol; Amersham, Arlington Heights, IL, U.S.A.) for 20 min, at 37°C sedimented at 18OOg for 15 min, washed successively with HEPES-Hanks buffer and finally resuspended in the buffer at a concentration of 2.5 x lo9 platelets per ml. Assays of serotonin release was routinely performed using 100 ~1 of platelet solution, 250 ~1 of plasma, 50 ~1 of HEPES-Hanks buffer, and 100 ~1 of the bacterial solution. Reactions were carried out in polystyrene tubes at 37°C for 30, 60 and 90 min, respectively. The tubes were then cooled to 4°C and centrifuged at 18OOg for 15 min. Supernatants were assessed for [3H]-serotonin and were expressed as a percentage of the total released
Rabbit platelet aggregation, release of ATP, and serotonin release induced by Strep. sanguis
and common serotypes of Strep. rabbit platelet aggregation and release of ATP as effectively as standard collagen. However, there were quantitative differences in the reactions elicited by these streptococci (Table 1). Strain 118-1, which was isolated from a patient with Behcet’s disease, induced the greatest aggregation/ ATP release ratio, and the shortest lag phase. Other isolates from patients with the disease (113-20 and 114-23) showed similar activities to those of the common serotypes. Serotonin release, after rabbit platelets were exposed to various serotypes of Strep. sanguis, are shown in Fig. 1. Uncommon serotypes induced greater release of serotonin than common serotypes; Both uncommon
sanguis induced
Table 1. Rabbit platelet aggregation and release of ATP by Strep. sanmis Strep. smguis Serotype
Strain
uncommon I 113-20 SSH-83 II KIH-T III 114-23 IV 118-l Common I ATCC 10556 II ATCC 10557 III ATCC 10558 IV ST-7 Collagen
Aggregation (%)
ATP release ratio”
Lag time (min)
54.3 * 17.0b 28.7 + 14.5* 21.3 + I3.5* 71.Ok22.1 93.7* 2.1**
0.82 + 0.37 0.35 & 0.17 0.30 & 0.26 3.64 f 2.23 4.505 1.44
2.0 * 0.5 3.3 * 0.3 2.4* 1.2 1.5*05 0.7 + 0.1
70.3 + 19.3 22.3 k 14.1* 53.3 f 11.7 67.0 + 11.4 60.3 &-11.2
0.17 f 0.08 0.56 f 0.15 0.94 10.45 3.06 + 1.59 1.00
1.9 * 1.2 2.2 _+0.3 2.1 f 0.6 1.5 + 0.5 1.0*0.5
aRatio of Strep. sanguis stimulation/collagen stimulation. bResults are given as mean (+ SD). *Significantly lower than % aggregation with collagen, p < 0.01. **Significantly higher than % aggregation with collagen, p < 0.01.
Platelet activation by Strep. sun@
427
human platelet-rich plasma. Strep. sanguis strains originally isolated from patients with Behqet’s or Kawasaki disease, unlike the common strains, promote platelet aggregation (Table 2). However, differences in the aggregation of platelet-rich plasma were observed among human donors. Responder frequency was 50% in patients with Behyet’s disease and 30% in controls. The levels of platelet aggregation at ATP release in responders are shown in Table 3. Aggregation in patients with Behyet’s disease was lower than that in healthy controls. However, aggregation induced by standard collagen was significantly higher in the patients (Table 4). Similar results were found for ATP release. Eflect of plasma from patients with Behget’s disease
0
30
Time
after
incubation
Plasma from non-responders with Behqet’s disease inhibited both aggregation and ATP release. The
90
60
15
(min)
Fig. 1. Serotonin release from rabbit platelets after stimulation with Sfrep. sanguk. 0: uncommon serotype I, upper, SSH-83, lower, 113-20; v: uncommon serotype II, KIH-T; A: uncommon serotype III, 114-23; m: uncommon serotype IV, 118-l; 0: common serotype I, ATCC 10556; V: common serotype II, ATCC 10557; A: common serotype. III, ATCC 10558; 0: common serotype IV, ST-7. a rapid increase
was found within
15 min, and after
30min the responses reached plateau. Human platelet aggregation and release at ATP induced by uncommon serotypes of Strep. sanguis
Strains of Strep. sanguis were tested for their abilities to promote platelet aggregation in fresh
inhibition was complete within 20min but over a longer period of observation, the platelet response could reappear. The levels of aggregation and ATP release were about half of those of the original levels. DISCUSSION
We show that only uncommon serotypes of Strep. platelets. Herzberg et al. (1985) have reported that Strep. sanguis is selectively recognized and bound by platelets, which respond physiologically. Surface component class I of Strep. sanguis mediates direct binding to a platelet-binding site (step 1). After adherence, the class II component of Strep. sanguis mediates Ca*+-dependent platelet
sanguis aggregate human
Table 2. Responsiveness of human platelets to Sfrep. sanguis
Platelets from
Subjects reacting with
Strep. smguis (%)
Responder ratio
113-20
SSH-83
114-23
118-l
Others”
s/10
40
40
10
20
0
10
30
10
10
0
Patients with Reh9et’s disease
NSb Healthy controls
3/10
aCommon serotypes I (ATCC 10556), II (ATCC 10557), III (ATCC 10558) and IV (ST-7). bNS: not significant. Table 3. Platelet aggregation and ATP release level in responders Strep. sanguis Platelets from
113-20
Patients with Behcet’s disease
Healthy controls
Aggregation (%) 16.0 k 13.4 Lag time (mm) 19.8 + 7.6 ATP release 0.13 f 0.10 ratio Aggregation (%) 40 Lag time (min) 11.5 ATP release
SSH-83
114-23
118-I
11.3 + 3.0 18.0 + 6.0 0.12 * 0.07
8 13
9.5 21
51.3 i24.8 11.3 * 1.2 0.19
14 17 -
22 15.8 -
Results are given as mean (k SD). Table 4. Platelet aggregation induced by collagen Platelets from Patients with Behcet’s disease Healthy controls *p <
Aggregation (%) ATP release level 83.0 f 6.3’ 65.9 f 13.9*
199.1 f 253.6* 65.9 f 13.9*
0.01; NS: not significant, unpaired two samples r-test.
Lag time (min) 0.97 & 0.34 NS 1.07 f 0.39 NS
428
EMIKOISOGAI
activation (step 2). The class III component may be an exo-ATPase that hydrolyses ATP (step 3). In our experiments, only clinical isolates (uncommon serotypes) activated human platelets, implying that there are distinct receptors for certain Strep. sang& strains on these cells. Selective platelet aggregation may contribute to the vasculitis occuring in Behget’s disease. Platelets contain a number of organelles that store polypeptides and low molecular-weight components. These organelles release their components after activating the clotting process. The a-granules containing plateletderived growth factor, @-thromboglobulin and fibrinogen. The primary in uiuo function of plateletderived growth factor is to induce mitosis in quiescent cells such as diploid fibroblasts and arterial smoothmuscle cells. This would corroborate the proposed in vivo role of this growth factor in wound healing. The ability of the factor to stimulate proliferation and migration of arterial smooth-muscle cells, and to stimulate synthesis of cholesterol esters prompted the suggestion that it might play a part in the pathogenesis of atherosclerosis (Ross and Glomset, 1976; Ross, 1981). In patients with Behvt’s disease, platelets that aggregated at the site of vascular injury may release the growth factor, which then initiates events involving ceI1 migrations and proliferation into the intima of arterial smooth-muscle cells. The process may lead to the formation of vascular lesions. About 30% of patients with Behget’s disease have vasculitis, and this may be related to platelet activation. We found that rabbit platelets were aggregated by all strains of Strep. sang&, although clinical isolates caused a greater release of serotonin than did the common strains. The differences in the results between serotonin release and aggregation/ATP release, may indicate methodological differences. The indirect method such as radiolabelling may be relevant only if non-thrombogenic surfaces are used and/or if platelet aggregation is inhibited (Sakariassen, Bolhuis and Sixma, 1979). From our experiments, we suggest that the origin of platelets and their bioassay are of importance in the investigation of the role of platelet abnormalities in Behvt’s disease. The coagulation system may augment immunologically mediated tissue injury in infections and autoimmune diseases (Naish, Penn and Evans, 1972; Vassalli and McCluskey, 1971). We found variation in both selectivity and rate of response among the platelet-rich plasmas from donors. Plasma from nonresponders delayed the platelet response of responders, indicating that plasma factors may influence the response. Such factors may be present in those patients with BehGet’s disease in whom the platelet response to the uncommon serotypes of Strep. sanguis was depressed, although the collagen-stimulated platelet response was significantly higher than in controls. Thus, the induction of Behcet’s disease may require infection with particular strains, or a mixture of Strep. sanguis, as well as the presence of appropriate receptors in platelets. In addition, plasma factors may effect the pathogenesis of this disease. Acknowledgemenr-These studies were supported by the Research Committee of Behqet’sdisease in Japan,
et al. REFERENCES
Barile M. F., Graykowsky E. A., Driscoll E. J. and
Rigges D. B. (1963) L form of bacteria isolated from recurrent aphthous stomatitis lesions. Oral Surg. 16, 139551402. Eglin R. P., Lehner T. and Subak-Sharpe J. H. (1982) Detection of RNA complementary to Herpes-simplex virus in mononuclear cells from patients with Behpt’s syndrome and recurrent oral ulcers. Luncet II, 1356-1360. Freedman L. R. and Valone J. Jr (1979) Experimental infective endocarditis. Prog. car&wax. Dis. 22, 169%180. Graykowski E. A., Barile M. F., Lee W. B. and Stanley H. R. (1966) Recurrent aphthous stomatitis. J. Am. med. Ass. 1%. 637644.
Herzberg M., C., Brinzenhofe K. L. and Clawson C. C. (1983) Aggregation of human platelets and adhesion of Streptococcus sanguis. Infect. Immun. 39, 1457-1469.
Herzberg M. C., Gong K., MacFarlane G. D., Ericson P. A., Soberay A. H., Krebsbach P. H., Manjula G., Schilling K. and Bowen W. H. (1990) Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis. Infect. Immun. 58, 512-522. Herzberg M. C., MacFarlane G. D. and Delzer P. R. (1985) Streptococcus sanguis interaction with human platelets. In Molecular Basis of Oral Microbial Adhesion (Eds Mergenhagen S. E. and Rosan B.)., RR. 53-60. American Society fo; Microbiology, U.S.A. __ Hook E. W. III and Sande M. A. (19741 Role of the vegetation in experimental Streptocdccus &idans endocarditis. fnfecf. Immun. 10, 1433-1438. Isogai E., Ohno S., Kotake S., Isogai H., Tsurumizu T., Fujii N., Yokota K., Syuto B., Yamaguchi M., Matsuda H. and Oguma K. (1990a) Chemiluminescence of neutrophils from patients with Behqet’s disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora. Archs oral Biol. 35, 4348.
Isogai E., Ohno S., Takeshi K., Yoshikawa Y., Tsurumizu T., Isogai H., Yokata K., Kotake S., Sasamoto Y., Hashimoto T., Shimizu H., Matsuda H., Fujii N., Yamaguchi M. and Oguma K. (1990b) Close association of Streptococcus sanguis uncommon serotypes with Behqet’s disease. BiJidobact. Microjora. 9, 27-24. Mayrand D. (1985)Virulence promotion by mixed bacterial infections. In The Pathonenesis of Bacterial Infections (Eds Jackson G. G. ad Thomas H.), pp. i81-291.
Springer, Berlin. Naish P., Penn G. B. and Evans D. J. (1972) The effect of difibrination on nephrotoxic serum nephritis in rabbits. Clin. Sci. 42, 643-646.
Ramirez-Ronda C. H. (1978) Adherence of glucan-positive and glucan negative streptococcal strains to normal and damaged heart valves. J. clin. Invest. 62, 805-814. Ramirez-Ronda C. H. (1980) Effect of molecular weight of dextran on the adherence of Streptococcus sanguis to damaged heart valves. Infect. Immun. 29, 1-7. Ross R. (1981) Atherosclerosis: a problem of the biology of arterial wall cells and their interaction with blood components. Atherosclerosis 1, 293-3 Il. Ross R. and Glomset N. (1976) The pathogenesis of atherosclerosis. New Engl. J. Med. 295. 369-376. Sakariassen K. S., Bolh& P. A., and Sixma J. J. (1979) Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium. Nature (Lond.) 279, 636638.
Scheld W. M., Valone J. A. and Sande M. A. (1978) Bacterial adherence in the pathogenesis of endocaiditis. J. din. Invest. 61, 1394-1404.
Platelet activation by Srrep. swguis The Beh@‘s Disease Research Committee of Japan (1989) Skin hypersensitivity to streptococcal antigens and the induction of systemic symptoms by the antigens in Behcet’s disease-a multicenter study. J. Reumatol. 16, 506-511. Vassalli P. and McCluskey R. T. (1971) The pathogenie role of the coagulation process in glomerular
429
disease of immunological origin. Adu. Nephrol. 1, 41-63. Yamanouch K., Shishido A., Kobune F., Sakota H. and Inaba G. (1981) Virological approaches to Behcet’s disease in Japan. In Behget’s Disease (Ed. Inaba G.), pp. 57-63. Univ. of Tokyo Press, Tokyo.