Embed Size (px)
Citation preview
Vol. 35, No. 1INFECTION AND IMMUNITY, Jan. 1982, p. 320-3250019-9567/82/010320-06$02.00/0
Effect of Nitrogen Mustard on Natural History of Right-SidedStreptococcal Endocarditis in Rabbits: Role for Cellular Host
DefensesBERTRAND R. YERSIN,j3 MICHAEL P. GLAUSER,' AND LAWRENCE R. FREEDMANI-2*
Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011Lausanne, Switzerland,1 and the Medical2 and Research3 Services of the Veterans Administration Wadsworth
Medical Center and the University of California at Los Angeles, California 90073
Received 15 May 1981/Accepted 4 September 1981
Cellular host defenses are considered to be ineffective in bacterial endocarditis;the microorganisms in infected vegetations are protected from phagocytic cells bydense layers of fibrin. To test this hypothesis, nitrogen mustard-induced agranulo-cytosis and leukopenia were produced in rabbits with right-sided streptococcalendocarditis. The spontaneous sterilization of tricuspid infection observed in thecontrol animals was not present in the granulocytopenic, leukopenic animals.Since the bacterium Streptococcus intermedius is not sensitive to the comple-ment-mediated bactericidal effect of serum and since the animals were notbacteremic during the time of agranulocytosis, an inhibitory effect of the drug on
local cellular host defense mechanisms is postulated. We suggest that thespontaneous sterilization of infective endocarditis in the right side of the heart inrabbits is mediated by cellular host defenses.
Since the development of the rabbit model otinfective endocarditis by Garrison and Freed-man in 1970 (13), many investigators have usedthe model to study the physiopathology of thisinfectious disease and its relationship to hostdefenses. Among other things, it has beenlearned from these studies that the complement-mediated bactericidal effect of serum is probablyan important mechanism protecting the hostagainst infection with serum-sensitive bacteria(2, 9, 17).The bacteria most commonly responsible for
infective endocarditis in humans are gram-posi-tive cocci. These microorganisms are resistantto complement-dependent serum bactericidalactivity, and infection, once established, isthought not to be affected by nonspecific orantibody-mediated phagocytosis (20). Neverthe-less, experiments with animals and gram-posi-tive bacteria have repeatedly demonstrated thatthe spontaneous sterilization of infective endo-carditis in the right side of the heart is a regularoccurrence (12). The mechanisms responsiblefor the sterilization of these infections have notbeen identified.The purpose of this study was to explore the
possible role of cellular host defense mecha-nisms in the spontaneous sterilization of right-sided infective endocarditis in rabbits. The ob-servation that dexamethasone inhibits thesterilization of this infection (12) suggested thathost cells might be important in this infection. In
these experiments, we examined the effect ofnitrogen mustard-induced agranulocytosis andleukopenia on the spontaneous sterilization oftricuspid valve-infective endocarditis due to astreptococcus resistant to complement-mediatedserum bactericidal activity.The tricuspid location of the streptococcal
infection was chosen since the rapid clearance ofbacteremia associated with this experimentalendocarditis permits the study of the local rela-tionship between host cells and infection with-out having to be concerned with the conse-quence of any experimental procedure on thefunction of bacterial clearance mechanisms out-side of the infected intracardiac vegetation.
MATERIALS AND METHODSProduction of bacterial endocarditis. New Zealand
white rabbits weighing, 1,500 to 2,800 g were operatedupon by the previously described technique (13) toproduce nonbacterial thrombotic endocarditis on thetricuspid valve. In brief, a polyethylene catheter wasintroduced into the right ventricle of the heart throughthe right jugular vein. Since the catheter was left inplace throughout the experiment, exact location of thetip of the catheter was possible at the time of sacrifice.Animals with misplaced catheters were not consideredin the study. Infection of nonbacterial thromboticendocarditis was realized by intravenous inoculationof 108 Streptococcus intermedius 6 to 7 days aftercatheterization.
Bacterial strain. A strain of S. intermedius originallyisolated from the blood of a patient with infective
320
on June 20, 2020 by guesthttp://iai.asm
.org/D
ownloaded from
TRICUSPID STREPTOCOCCAL ENDOCARDITIS
endocarditis and previously used in other studies (12,15) was used. This bacterium is known to be resistantto the bactericidal effect of rabbit serum. After over-night culture at 37°C in tryptone soy broth (DifcoLaboratories, Detroit, Mich.), the bacteria were sus-pended in normal saline to obtain a standard inoculumequal to 108 colony-forming units (CFU) per ml. Thisinoculum was enumerated by plating serial dilutionson blood agar medium.
Evaluation of infection. Blood was cultured at differ-ent intervals between bacterial challenge and sacrificeof the animals. For this purpose, blood was obtainedby puncturing the central artery of the ear, and 1 ml ofblood was immediately cultured on blood agar plates.At the time of sacrifice, animals were killed with an
overdose of pentobarbital administered intravenously.Tricuspid vegetations, spleens, and left kidneys wereremoved, weighed, and ground; serial dilutions ofhomogenates were cultured quantitatively on bloodagar plates. The bacterial counts were expressed asloglo CFU per gram of tissue.
Histological studies. At the time of sacrifice, sometricuspid vegetations were divided into two parts tostudy the histological structure of the vegetations. Onepart of these vegetations was fixed in a formaldehydesolution (37% formaldehyde; Merck & Co., Inc.,Darmstadt, Germany) and then prepared and stainedwith hematoxylin-eosin and by the method of Gram.
Drug-induced leukopenia. Six days after bacterialchallenge, 24 rabbits received intravenously 2.5 mg ofnitrogen mustard (mechlorethamine, Cloramin; Simes,Milano, Italy) per kg. Simultaneously, a blood culturewas taken from each of these animals to verify thatnone were bacteremic at the time of drug administra-tion.At the time of nitrogen mustard (HN2) administra-
tion and each day until the sacrifice of the animals,blood was removed by arterial puncture so that aleukocyte count (Coulter Counter model Dn; CoulterElectonics Ltd., Dunstable, Great Britain) and a poly-morphonuclear leukocyte (PMN) count after stainingof a blood smear with a May-Grunwald-Giemsa staincould be performed.
Nitrogen mustard was also given at the same dosageto 10 normal rabbits. Leukocyte and PMN countswere carried out in the same way to determine thehematological profile of drug-induced leukopenia innoninfected animals.Groups of animals. The group of 24 experimental
animals received 2.5 mg of HN2 per kg intravenously 6days after the bacterial challenge and were sacrificed 5days later (at day 11).Among the 17 control animals, 5 were sacrificed at
day 6. At that time, the remaining 12 rabbits receivedan injection of normal saline and were sacrificed 5days later (at day 11).
Statistical evaluation. The vegetations in the differ-ent groups were compared with the x2 test (with theYates correction).The mean number of bacteria (expressed as loglo
CFU/g of vegetations) in tricuspid vegetations werecompared among the different groups with the Studentt test. In these comparisons, three different methodswere used to calculate the mean: (i) sterile vegetationswere included (counted as loglo CFU/g = 2 since theweight of vegetations is approximately 0.1 g), (ii) onlyvegetations of nonbacteremic animals at the time of
sacrifice were considered (iii) only infected vegeta-tions were considered.
RESULTS
Natural history of the disease. Six days afterbacterial challenge, all five sacrificed rabbits hadinfected tricuspid vegetations: x log10 CFU/g =6.52 (Table 1).
Five days later (at day 11) among the 12sacrificed animals, only 10 could be evaluated(because two catheters were misplaced). Of therabbits, 5 of 10 had infected vegetations: x logl0CFU/g = 3.77 (n = 10). Blood cultures betweendays 2 and 11 were always sterile, except for onefrom an animal at day 11. Cultures of left kid-neys and spleens at day 6 or 11 remained sterilein all animals except for one spleen (log10 CFU/g= 3.81) in the bacteremic animal at day 11.Drug-induced leukopenia. On day 2 after the
administration of nitrogen mustard to 10 normalrabbits, a severe leukopenia associated withagranulocytosis (PMN count, <100 cells permm3) developed and persisted for 48 h (Fig. 1).Hematological improvement began 6 days afterthe administration of the drug, and the leukocyteand PMN counts were almost normal 24 h later.
In the 24 infected rabbits, the hematologicalprofiles were almost the same after the adminis-tration of nitrogen mustard, and agranulocytosiswas present for at least 48 h (Fig. 1).
Effect of nitrogen mustard-induced leukopeniaon the natural history of the disease. Twenty-fourrabbits received an injection of nitrogen mustard(2.5 mg/kg intravenously) 6 days after the induc-tion of infective endocarditis. Blood culturestaken at the same time in these 24 animals weresterile.Four animals died spontaneously in the days
after drug administration; among these four rab-bits, three had vegetations infected with gram-negative bacteria. At time of sacrifice (day 11),five other animals had a systemic gram-negativesuperinfection.Because of the misplacement of the catheter
(inferior vena cava), two other animals wereexcluded from the study.Among the 13 rabbits considered for evalua-
tion in the group given nitrogen mustard, 11 hadinfected vegetations, and 3 of them had positiveblood cultures at the time of sacrifice. Spleensand left kidneys were always sterile except inthe three bacteremic animals.The progressive spontaneous sterilization ob-
served in the controls was not present in theanimals that had received nitrogen mustard evenif the data were considered excluding bacter-emic animals (Table 1; Fig. 2). In other words,the leukopenia and agranulocytosis present in
321VOL. 35, 1982
on June 20, 2020 by guesthttp://iai.asm
.org/D
ownloaded from
TABLE 1. Natural history of tricuspid streptococcal infection in rabbits and the effect of nitrogen mustard-induced leukopeniaa
No. of infected No. of Log1o CFU/g of vegetations (mean t SEM)
Expt group vegetations/total bacteremic b Nonbacteremic Animals with
rabbits anilmals animalsc infected vegetations'Control animals at day 6 5/5 0 6.52 + 0.82 6.52 ± 0.82 6.52 ± 0.82Control animals at day 11 5/10 1 3.77 ± 0.78e 3.18 ± 0.58e 5.59 ± 1.08HN2-treated animals atday 11 11/13 3 6.89 ± 0.71f 6.25 ± 0.82f 7.78 ± 0.45f
a Only animals that could be evaluated at the time of sacrifice were considered in this table.b Sterile vegetations were included in the mean as log10 CFU/g = 2.Only vegetations of nonbacteremic animals at the time of sacrifice were considered.
d Only infected vegetations were considered.e Significantly different (P, <0.05; Student t test) from control animals at day 6.f Significantly different (P, <0.05; Student t test) from control animals at day 11.
these rabbits seem to have interfered with theability of the host to sterilize the tricuspid infec-tion. If we consider the unlikely possibility thatsterile animals in all groups may represent ani-mals which did not become infected at the timeof bacterial challenge (despite the 100% infec-tion in the rabbits sacrificed at day 6), and wethus remove sterile vegetations from the meansof bacterial counts, no significant sterilization isobserved in the natural history of the disease(Table 1). Nevertheless, in the granulocytopenicleukopenic animals, a significant increase in thebacterial counts of infected vegetations wasseen.
Histological structure of vegetations. The mi-croscopic examination of tricuspid vegetations
z 90008 S
o 8000 \
0 7000 \0
E 6000-E
z E \_jv000500
w 000
U0 A A2000-
w A
-' 1000- NH2 I-V
1 2 3 4 56 7DAYS
FIG. 1. Effect of nitrogen mustard administrationupon the mean leukocyte (0) and PMN (A) counts in10 uninfected rabbits ( ) and in 24 rabbits with rightstreptococcal endocarditis (---).
demonstrated the usual structure of fibrin andplatelets. Bacterial colonies were seen onlywhen the bacterial count of the vegetation waslog10 CFU/g > 7 and when present, appeared asfoci apparently isolated from inflammatory cellsby fibrin layers. But, even in granulocytopenicrabbits, the content of inflammatory cells in thevegetations appreciated by the number of cellsin the microscope field was sometimes impor-tant (Fig. 3).
DISCUSSIONSince the study of the rabbit model of endo-
cardial infection, it has been demonstrated thatexperimental tricuspid infections have a naturaltendency to evolve toward spontaneous steril-ization, whereas aortic endocarditis is always asevere disease almost uniformly fatal (2, 11, 12,
CONTROL GROUPS GROUP NH2
p<O.OIpcO.OFI
Uf)z0
w
v
w
N,
LL.
0
DAY 6 DAY II DAY 11
FIG. 2. Effect of nitrogen mustard-induced leuko-penia and agranulocytosis on the natural history ofright heart streptococcal endocarditis in rabbits. HN2was administered on day 6. Only animals with sterileblood cultures were considered in this figure. Eachpoint represents one animal. Bars represent meanvalues. See text for details of calculation.
322 YERSIN ET AL. INFECT. IMMUN.
on June 20, 2020 by guesthttp://iai.asm
.org/D
ownloaded from
324 YERSIN ET AL.
19). Furthermore, it has recently been shownthat other experimental intravascular infectionslocated in the thoracic or abdominal aorta andthe inferior vena cava also have a natural ten-dency toward rapid sterilization (12). In otherwords, host defense mechanisms are effective inintravascular infections in all locations testedwithin the vascular system, except, perhaps, forinfections of the aortic valve. The differencesbetween left-sided and right-sided endocarditisand other infections located in the vascularsystem are not understood but clearly cannot beexplained only by differences in local P02, bloodpressure, or hemodynamic factors in the differ-ent intravascular sites. Furthermore, dexa-methasone treatment of rabbits has no effect onleft-sided streptococcal endocarditis, whereasthe spontaneous sterilization of right-sided in-fection is totally inhibited (12).Our study has shown that nitrogen mustard
modifies the natural history of right-sided strep-tococcal endocarditis in rabbits. The naturaltendency of this infection toward spontaneoussterilization was absent in leukopenic rabbits. Ofparticular importance is the absence of bacter-emia during the time of leukopenia and agranulo-cytosis, thus rendering highly improbable anysystemic effect of the drug on circulating bacte-ria and thereby supporting the view that there isa local effect of cells upon the evolution ofinfection within certain intravascular infections.The microscopic structure of these experi-
mentally induced intravascular vegetations hasbeen reported by many authors and appears tobe a kind of meshwork of fibrin and plateletswhere inflammatory cells are sparse. Further-more, the bacteria appear to be situated in thevegetations, apparently protected from phago-cytic cells, which are said to be unable topenetrate fibrin (6-8, 10, 12, 13). No differencesin histology have been reported between left-and right-sided infections. On the other hand,we have observed several tricuspid vegetationsin which inflammatory cells seemed numerous, afinding consistent with some descriptions oftricuspid endocarditis in humans in which PMNswere consistently present on histological exami-nations and often produced abscesses in thevegetations (22).The differences observed in the natural his-
tory of different intravascular infections in theanimal model are reflected in the more favorableresponse to antibiotic treatment and thus theprognosis in right-sided staphylococcal or Pseu-
domonas endocarditis compared with left-sidedinfection (1, 21). Furthermore, in a recent re-view of the treatment of infective endocarditis,the mortality was 35% in 59 cases of left-sidedstaphylococcal endocarditis, whereas only 13%of patients died in 72 cases of right-sided in-volvement (4).Thus, the bacteriological response to antibiot-
ic treatment seems to be better in the right sideof the heart, and, since there is evidence thatphagocytic cells act synergistically with antibiot-ics to kill bacteria, this response is a furtherargument suggesting that cellular host defensesprobably play a role in intravascular infections,at least in tricuspid endocarditis.There is little to suggest that interference with
any complement-dependent lytic system wasresponsible for the effect of nitrogen mustard.All gram-positive cocci are resistant to lysis bycomplement (3, 5). A recent study of immuniza-tion on the induction and course of Streptococ-cus sanguis endocarditis in rabbits showed noeffect of preimmunization on the bacterial con-tent of the vegetations (23). Furthermore, de-spite increased rates of phagocytosis of S. san-guis in the presence of immune serum in vitro,there was no increase in intracellular killing ofthese preopsonized streptococci. Finally, in vi-tro growth of streptococci in 10% immune serumwas no different from that in control serum (23).We have, in addition, incubated 103 strepto-
cocci with 90% normal rabbit serum for 24 h,and no bacterial killing was observed (B. Yersinand L. R. Freedman, unpublished data).Thus, even if the effect of nitrogen mustard
were mediated by virtue of its decreasing anti-body production, it would appear to be highlyunlikely that this would decrease a hypotheticallytic system which has never been demonstrat-ed. Rather, it is much more reasonable to relatethe effect of nitrogen mustard to (i) a decrease inthe number of effector cells, (ii) decreased opso-nin, thereby interfering with phagocytosis, or(iii) decreased antibody, thereby interfering withthe generation of a chemotactic factor whichpresumably would affect the course of bacterialinfection through the intermediary of a cellularelement.When serum killing of gram-positive cocci has
been observed in vitro, it has been found to beattributable to the release in the serum of plate-let bactericidal substances (16), previous expo-sure of bacteria to subinhibitory concentrationsof antibiotics (18), or an unknown phenomenon
FIG. 3. (A) Histological section of a tricuspid valve vegetation from a rabbit challenged 11 days before with S.intermedius and spontaneously sterilized. There are numerous inflammatory cells inside the vegetation.Hemotoxylin-eosin stain (x75). (B) Section of an 11-day-old infected tricuspid vegetation in a granulocytopenicrabbit. Bacterial colonies appear isolated from the sparse inflammatory exudate. Hematoxylin-eosin stain (x75).
INFECT. IMMUN.
on June 20, 2020 by guesthttp://iai.asm
.org/D
ownloaded from
TRICUSPID STREPTOCOCCAL ENDOCARDITIS
not dependent upon the presence of antibody orcomplement (14).With all of the differences of opinion concern-
ing the pathophysiology of infective endocardi-tis, the absence of a role of cellular host defensesseems to have been generally accepted. Thesestudies question this generally accepted view.They do not provide any information, however,concerning the nature of the cell(s) which isimportant or the mechanism by which the anti-bacterial effect is mediated. Since microbial col-onies in endocardial vegetations are separatedby layers of fibrin from those leukocytes whichare occasionally seen in histological sections,these studies raise the possibility that leukocytescontribute to antibacterial host defenses bymechanisms other than those of direct contactand phagocytosis.
ACKNOWLEDGMENTS
This work was supported by grants from the SICPA Foun-dation, Lausanne, Switzerland, the Intermaritime Founda-tion, Geneva, Switzerland, and the Research Service of theVeterans Administration, Los Angeles, Calif.The technical assistance of M.-N. Gay-Balmaz and M.
Ramseyer is gratefully acknowledged.
LITERATURE CITED
1. Abrams, B., A. Sklaver, T. Hoffman, and R. Greenman.1979. Single or combination therapy of staphylococcalendocarditis in intravenous drug abusers. Ann. Intern.Med. 90:789-791.
2. Archer, G., and F. R. Fekety. 1976. Experimental endo-carditis due to Pseudomonas aeruginosa. I. Descriptionof a model. J. Infect. Dis. 134:1-7.
3. Braude, A. I. 1981. Mechanisms of resistance to infection,p. 739-764. In A. I. Braude (ed.), Medical microbiologyand infectious diseases. The W. B. Saunders Co., Phila-delphia, Pa.
4. Bryant, R. E., and R. C. Kimbrough. 1978. Treatment ofinfective endocarditis, p. 327-360. In S. H. Rahimtoola(ed.), Infective endocarditis. Grune & Stratton, Inc., NewYork.
5. Davis, B. D., R. Dulbecoo, H. N. Eisen, H. S. Ginsberg,and W. B. Wood, Jr. 1968. Host-parasite relations inbacterial diseases, p. 603-651. In B. D. Davis, R. Dulbe-coo, H. N. Eisen, H. S. Ginsberg, and W. B. Wood (ed.),Microbiology. Harper & Row, Publishers, New York.
6. Durack, D. T. 1975. Experimental bacterial endocarditis.IV. Structure and evolution of very early lesions. J.Pathol. 115:81-89.
7. Durack, D. T., and P. B. Beeson. 1972. Experimental
bacterial endocarditis. I. Colonization of a sterile vegeta-tion. Br. J. Exp. Pathol. 53:44-49.
8. Durack, D. T., and P. B. Beeson. 1972. Experimentalbacterial endocarditis. II. Survival of bacteria in endocar-dial vegetations. Br. J. Exp. Pathol. 53:50-53.
9. Durack, D. T., and P. B. Beeson. 1977. Protective role ofcomplement in experimental Escherichia coli endocardi-tis. Infect. Immun. 16:213-217.
10. Durack, D. T., and P. B. Beeson. 1978. Pathogenesis ofinfective endocarditis, p. 1-53. In S. H. Rahimtoola (ed.),Infective endocarditis. Grune & Stratton, Inc., NewYork.
11. Durack, D. T., P. B. Beeson, and R. G. Petersdorf. 1973.Experimental bacterial endocarditis. III. Production andprogress of the disease in rabbits. Br. J. Exp. Pathol.54:142-151.
12. Francioli, P. B., and L. R. Freedman. 1979. Streptococcalinfection of endocardial and other intravascular vegeta-tions in rabbits: natural history and effect of dexametha-sone. Infect. Immun. 24:483-491.
13. Garrison, P. K., and L. R. Freedman. 1970. Experimentalendocarditis. I. Staphylococcal endocarditis in rabbitsresulting from placement of a polyethylene catheter in theright side of the heart. Yale J. Biol. Med. 42:394-410.
14. Guze, L. B., E. G. Hubert, and G. M. Kalmanson. 1970.Pyelonephritis. XI. Effect of growth phase of Streptococ-cus faecalis on serum susceptibility of virulence. Infect.Immun. 1:532-537.
15. Heraief, E., M.-P. Glauser, and L. R. Freedman. 1980.Vancomycin prophylaxis of streptococcal endocarditis inrats, p. 911-913. In J. D. Nelson and C. Grassi (ed.),Current chemotherapy and infectious diseases, vol. II.
American Society for Microbiology, Washington, D.C.16. Hirsch, J. G. 1960. Comparative bactericidal activities of
blood serum and plasma serum. J. Exp. Med. 112:15-22.17. Kaspar, R. L., and D. J. Drutz. 1977. Perihepatitis and
hepatitis as complications of experimental endocarditisdue to Neisseria gonorrhoeae in the rabbit. J. Infect. Dis.136:37-42.
18. Lorian, V., and B. Atkinson. 1978. Effect of serum ongram-positive cocci grown in the presence of penicillin. J.Infect. Dis. 138:865-871.
19. Perlmann, B. B., and L. R. Freedman. 1971. Experimentalendocarditis. III. Natural history of catheter inducedstaphylococcal endocarditis following catheter removal.Yale J. Biol. Med. 44:214-224.
20. Phair, J. P., and J. Clarke. 1979. Immunology of infectiveendocarditis. Prog. Cardiovasc. Dis. 22:137-144.
21. Reyes, M. P., W. A. Palutke, R. F. Wylin, and A. M.Lerner. 1973. Pseudomonas endocarditis in the DetroitMedical Center, 1969-1972. Medicine (Baltimore) 52:173-194.
22. Roberts, W. C., and N. A. Buchbinder. 1972. Right-sidedvalvular infective endocarditis. A clinico-pathologic studyof twelve necropsy patients. Am. J. Med. 53:7-19.
23. Thorig, L., J. Thompson, and R. van Furth. 1980. Effect ofimmunization on the induction and course of experimentalStreptococcus sanguis and Staphylococcus epidermidisendocarditis. Infection 8:267-274.
VOL. 35, 1982 325
on June 20, 2020 by guesthttp://iai.asm
.org/D
ownloaded from
