Jun 25, 1976 - philus influenzae type b septicemia and meningitis. ..... John Wiley and Sons, New York. 5. ... Newman, S. L., B. Waldo, and R. B. Johnston, Jr.
Vol. 14, No. 4 Printed in U.S.A.
INFECTION AND IMMUNITY, Oct. 1976, p. 882-887 Copyright C 1976 American Society for Microbiology
Participation of Complement in the Nonimmune Host Defense Against Experimental Haemophilus influenzae Type b Septicemia and Meningitis FRANCIS J. CROSSON, JR.,* JERRY A. WINKELSTEIN, AND E. RICHARD MOXON Departments ofPediatrics* and Microbiology, The Johns Hopkins University School ofMedicine, Baltimore, Maryland 21205 Received for publication 25 June 1976
This study was undertaken to determine whether the terminal complement components (03-9) are involved in the nonimmune host defense against Haemophilus influenzae type b septicemia and meningitis. Using cobra venom factor, infant rats were depleted of C3 and C5. After intranasal challenge with H. influenzae type b, the complement-depleted rats developed a greater incidence and magnitude of bacteremia and a higher mortality rate. In contrast to the effects on bacteremia, complement depletion did not directly influence either the occurrence of meningitis or bacterial multiplication within the cerebrospinal fluid. These experiments provide evidence that the complement system may be an important mechanism of natural immunity to H. influenzae type b.
Haemophilus influenzae type b (HITB) is a significant cause of septicemia and meningitis in infants and young children (16). There is substantial evidence that anticapsular antibody plays an important role in acquired immunity to HITB, but protective titers of this antibody are rarely present in the blood of young children (17). Therefore, other host defense mechanisms must play a role in the prevention of HITB infections in young children. However, such mechanisms of natural immunity to HITB have not been well defined. Studies performed in vitro have shown that activation of the terminal complement components (03-9) by HITB can result in opsonization of the organism (11). However, it is not known whether the activation of C3-9 is an important in vivo defense mechanism against HITB. It has been demonstrated previously that infant rats, lacking anticapsular antibody, will develop bacteremia and meningitis after intranasal inoculation with HITB (10). The availability of this animal model has made possible the present studies, which were undertaken in order to investigate the role of the terminal complement components in the nonimmune host defense against HITB directly and in vivo. MATERIALS AND METHODS Animals. Newborn COBS/CD Sprague-Dawley rats were purchased from Charles River Laboratories, Wilmington, Mass., and maintained as previously described (10). 882
Cobra venom factor. Lyophilized Naja naja venom was purchased from Ross Allen Reptile Institute, Silver Spring, Fla. Cobra venom factor (CoVF) was purified as previously described (13); the purified CoVF gave a single band on polyacrylamide gel electrophoresis. After purification, the CoVF was divided into aliquots and frozen at - 70°C until used. C3 and C5 determinations. Serum was obtained from groups of five to ten rats by serial tail bleeding, pooled, and stored at -70°C. C3 and C5 titers were determined simultaneously on all samples, using hemolytic assays (14, 15). Bacterial growth, inoculation, and quantitation. The strain used was a streptomycin-resistant mutant of HITB (Eagan), which was grown as previously described (10). The animals were inoculated intranasally (10) with the desired number of logphase organisms suspended in 0.01 ml of phosphatebuffered saline. The concentration of bacteria was determined spectrophotometrically by using a Lumitron colorimeter 401 (Photovolt Corp., New York) and confirmed the next day by viable count on solid media. For serial quantitative blood cultures, 0.01ml samples of tail blood were obtained with a sterile micropipette and plated directly and at a 1:100 dilution on solid media containing 500 ,ug of streptomycin (Pfizer Corp., New York) per ml. Colonies were counted at 24 h, and the results were expressed as colony-forming units (CFU) per milliliter ofblood. CSF collection. Cerebrospinal fluid (CSF) was collected by a modification of a previously described method (9). Briefly, the animals were anesthetized with 40 mg of sodium pentobarbital (Abbott Laboratories, Chicago) per kg, and the tissues at the base of the skull were separated to reveal the dura overlying the cisterna magna. This was cleaned free of blood, and the cisterna was entered by direct puncture with a very finely drawn-out Pasteur pipette.
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About 20 ,ul of uncontaminated CSF could regularly be obtained in this way. The leukocyte concentration was determined by using a hemocytometer and expressed as cells per cubic millimeter. Methylene blue-stained preparations were used to determine the relative proportion of polymorphonuclear to mononuclear cells. Appropriate dilutions were cultured on solid media, read the next day, and expressed as CFU per milliliter of CSF. Statistical analysis. The significance of frequency distribution differences was determined by using the x2 test with the Yates continuity correction (4). Sample means were compared by using a two-sample t test (4).
RESULTS Effect of CoVF on serum C3 and C5 titers. Ten-day-old infant rats were injected intraperitoneally with either 25 ug of CoVF in 0.25 ml of sterile saline or 0.25 ml of sterile saline alone. The serum C3 titer fell to near zero in the CoVF-treated group and remained so until day 6, when the titer began to increase. C3 titers in control animals increased over the 6 days, presumably reflecting the ontogeny of C3 in the growing infant rat (Fig. 1A). The serum C5 titer fell to about 50% of the initial titer in the CoVF-treated animals and remained in that range for 4 days, increasing to a near-normal titer on day 5 (Fig. 1B). There was a moderate increase in the C5 titer of control animals during the same period. There were no apparent ill effects or mortality among the animals in either group. Thus, animals treated with CoVF became markedly deficient in C3 and moderately deficient in C5 for a period of 4 to 5 days, during which time their response to HITB intranasal challenge was examined. Effect of complement depletion on the incidence and magnitude of bacteremia. To study the effect of complement depletion on the incidence and magnitude of HITB bacteremia, littermates were given either CoVF or saline, as above, and inoculated intranasally with HITB, and the subsequent occurrence of bacteremia was determined. The peak cumulative incidence of bacteremia in animals given 4 x 106 HITB was 12 of 19 (63%) of CoVF-treated animals and 5 of 20 (25%) of controls (X2c = 4.32; P < 0.05) (Fig. 2). In animals given 2 x 107 HITB, the peak incidence was 18 of 19 (95%) of CoVF-treated and 13 of 19 (63%) of control rats (X2c = 2.30; P < 0.10). All animals who became bacteremic remained bacteremic during the course of the study. Thus, although the incidence of bacteremia was dependent on the size of the HITB inoculation, it was always higher among the complementdepleted animals and significantly higher
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FIG. 1. Effect of CoVF treatment of 10-day-old rats on (A) serum C3 titer and (B) serum C5 titer. The arrows signify the injection of either 0.25 ml of saline (*) or 25 pg of CoVF in 0.25 ml of saline (A).
among the complement-depleted animals given the lower inoculum. Blood from animals inoculated with 2 x 107 HITB was also used to determine the magnitude of bacteremia in each animal. Geometric mean bacteremia (mean log,0 CFU per milliliter) was consistently greater in the CoVFtreated group than in the control group (Fig. 3). This difference was greatest at days 3, 4, and 5 and represented a 20- to 50-fold greater bacteremia in the CoVF-treated group (P < 0.001 for days 3, 4, and 5). In most individual control animals, bacteremia increased to 104 to 105 CFU/ml over 2 to 3 days and plateaued at that level. In individual CoVF-treated animals, bacteremia increased to 105 to 10c in 0 to 1 days. Bacteremia greater than 2.0 x 106 CFU/ml was always followed within 1 day by death of the animal (see below). The observed decrease in geometric mean bacteremia among CoVFtreated animals on day 6 reflected the mortality that had occurred in this group at that time rather than a decrease in the magnitude of
INFECT. IMMUN. CROSSON, WINKELSTEIN, AND MOXON A A A A oo rats not only developed a greater magnitude of E 90bacteremia but also had a higher mortality rate 4' 80than did control animals. 0 Effect of complement depletion on the inci/0dence and characteristics of meningitis. To A 60study the occurrence of meningitis among bac0. 50teremic complement-depleted animals as compared with bacteremic control animals, infant 40wA Irats were given either CoVF or saline and inoc30ulated as previously with 2 x 107 HITB. The A number of control animals was increased in 20/ I 0 order l o to yield in about o o equalSerial number of bacter/ o-~ emic animals eachangroup. F quantitative i blood cultures were obtained, and on day 3 or 4 Days after inoculation CSF was collected from bac'IG. 2. Cumulative incidence of bacteremia teremic animals. Meningitis was defined by a amcrng CoVF-treated infant rats (A) and control rats Positive culture of the uncontaminated CSF at (0) inoculated intranasally with 4 x 106 HITB, and 24 h. Two animals had meningitis without CSF am ong CoVF-treated infant rats (A) and control rats pleocytosis, and meningitis never occurred in animals without bacteremia. (0) inoculated intranasally with 2 x 107 HITB. As depicted in Fig. 5, 18 of 26 (69%) CoVF7 treated bacteremic rats and 12 of 26 (46%) controls had meningitis. However, geometric mean bacteremia was 1.2 x 106 CFU/ml in A 6 CoVF-treated rats and 1.8 x 105 CFU!ml in 4' / \ control rats. The higher incidence of meningitis A -i / among CoVF-treated rats was directly related m D to the greater number of animals having a U.4 . greater magnitude of bacteremia in that group. A/ .-*----. 4O Thus, among animals with bacteremia greater 4 than 5 x 104 CFU/ml, 18 of 20 CoVF-treated and 11 of 11 controls had meningitis. Among 3with bacteremia less than 5 x 104 animals 0 CFU/ml, none of 6 CoVF-treated and 1 of 15' E0 controls had meningitis. Thus, there was a div4 w 2 2 3 4f 5 6 rect relationship between the presence of bac1 2 3 4 6 teremia greater than 5 x 104 CFU/ml and the LJUtb uays
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FIG. 3. Geometric mean bacteremia (log,0 CFU per milliliter) among infant rats treated with CoVF (A) or saline (a) and inoculated intranasally with 2 x 107 HITB.
bacteremia in surviving animals. Thus, the complement-depleted animals developed a significantly greater magnitude of bacteremia than did control animals. Effect of complement depletion on mortality. Beginning with day 3 of the above experiments, deaths occurred among the CoVFtreated rats. All animals who died had HITB bacteremia. There were no deaths among control animals receiving either HITB inoculum dose (Fig. 4). However, the cumulative mortality among CoVF-treated rats inoculated with 4 x 106 HITB was 9 of 19 (47%) (X2c = 9.79; P < 0.01) and among CoVF-treated rats inoculated with 2 x 107 HITB was 13 of 19 (68%) (X2c = 16.8; P < 0.001). Thus, complement-depleted
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presence of meningitis, and this "threshold" for meningitis was not altered by complement depletion. Quantitative CSF bacterial counts ranged from 1.0 x 102 to 4.0 x 108 CFU/ml (geometric mean of 1.4 x 106 CFU/ml) in control animals and from 1.0 x 102 to 4.0 x 108 CFU/ml (geometric mean of 8.9 x 105 CFU/ml) in CoVFtreated animals (Fig. 6A). The difference was not significant (P > 0.80). Thus, although the complement-depleted rats had a significantly greater magnitude of bacteremia than did control rats, bacterial counts in the CSF were equivalent in both groups. Therefore, although complement-dependent mechanisms limited the magnitude of bacteremia, they appeared
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not to affect the multiplication of the bacteria
in the CSF. CSF leukocyte counts ranged from 50 to 24,000 cells/mm3 (mean, 11,050 cells/mm3) in control animals and from 0 to 9,600 cells/mm3 (mean, 2,626 cells/mm3) in CoVF-treated animals. This difference was highly significant (P < 0.001). Differential counts were similar for both groups, showing 60 to 70% polymorphonuclear and 30 to 40% mononuclear cells. Thus, although CSF bacterial counts were similar for both groups, the number of leukocytes was more than fourfold greater in control animals than in complement-depleted animals.
DISCUSSION HITB is a significant cause of septicemia and _ 7 LA meningitis in infants and young children. Since . these infants rarely possess anticapsular antiAA body against HITB, the mechanisms by which U 6such nonimmune hosts defend against infec2 AA 00 tions by this organism are unclear. .000 -J5 The complement system appears to have a in the protection of the nonimmune host role E0 from some bacterial infections. Opsonizing, chemotactic, anaphylatoxic, and bactericidal m activities have been attributed to products of 00e the terminal complement components (C3-9) 0 00 (7, 12, 18). This sequence may be activated via the classical complement pathway in the preso ence of even small amounts of antibody or via the alternative or properdin pathway, which C oVF TREATED CONTROL may function independent of antibody and FIG. 5. Relationship between the magnitude of therefore be more important in the pre-antibacteremia (log,0 CFU per milliliter) and the pres- body phase of host defense (18). In recent years, ence (closed symbols) or absence (open symbols) of meningitis in infant rats treated with CoVF or sa- patients have been studied who have increased line, inoculated intranasally with 2 x 107 HITB, and susceptibility to pyogenic infections associated with abnormalities of the terminal complement sacrificed at 3 to 4 days for CSF examination. 0
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FIG. 6. Cerebrospinal fluid findings in meningitis ofinfant rats treated with either CoVF (A) or saline (0), inoculated with 2 x 107 HITB, and sacrificed at 3 to 4 days for CSF examination. (A) Bacterial counts (log,0 CFU per milliliter); (B) leukocyte counts (cells per cubic millimeter).
886
INFECT. IMMUN.
CROSSON, WINKELSTEIN, AND MOXON
components (1-3, 8). It has also been shown that C5-deficient and C3-depleted mice have increased susceptibility to pneumococcal (19) and staphylococcal (5) infections. The present study was designed to determine whether C3-9 is involved in the nonimmune host defense against HITB septicemia and meningitis, and, if so, to delineate where in the pathogenesis of the infections C3-9 exerts its protective effects. Using CoVF, infant rats were depleted of C3 and rendered moderately deficient in C5. Since C3 is required for activation of the terminal complement cascade, C3 depletion should render the animal functionally depleted of all C39-mediated activities (1). The demonstration of a different response to HITB challenge in the complement-depleted animals would, therefore, provide insight into the protective effects of these complement-related activities in the intact nonimmune host. The experimental model of HITB septicemia and meningitis used in these studies was one that closely resembles, in the route of entry, spread of organisms, and histopathologic findings, the disease in humans
tic activity due to C5 depletion as well as decreased 03-dependent C5 activation. Such complement-related chemotactic activity may be important in the earli'est stages of the inflammatory response and hence may be involved in the early entry of leukocytes into the CSF. Alternatively, the lower CSF leukocyte counts in the CoVF-treated animals could reflect a depressed peripheral leukocyte response due to overwhelming bacteremia in these animals. Determinations of blood leukocyte counts in bacteremic CoVF-treated animals and controls, however, did not support this latter hypothesis. HITB is a major pathogen of young children. Acquired immunity to HITB is believed to require production of anti-polyribophosphate antibodies by the host, and this generally does not occur below the age of 2 years. Other mechanisms, therefore, must play a role in the prevention of serious infections by this organisms in young children. These experiments provide evidence that the complement system may be an important mechanism of such natural immunity to HITB.
(10). ACKNOWLEDGMENTS After bacterial challenge, the complementWe would like to thank David Carver for his review of depleted rats developed a greater incidence and magnitude of bacteremia and a higher mortal- this manuscript and Camille Carruth and Susan Kinkel for assistance. ity rate than did controls. Such differences secretarial This work was supported by Public Health Service could reflect an inability of the complement- grants AI-00461 and AI-11637 from the National Institute of depleted animals to contain the bacteria at the Allergy and Infectious Diseases and NS-12554 from the initial site of inoculation, the nasopharynx, National Institute of Neurological Diseases and Stroke and the Hospital for Consumptives of Maryland (Eudowood), and/or an inability to clear bacteria from the by Baltimore. Jerry Winkelstein is an investigator of the Howbloodstream. All control animals limited the ard Hughes Medical Institute. magnitude of their bacteremia to nonlethal levels. Most complement-depleted animals, on the LITERATURE CITED other hand, developed a greater magnitude of 1. Alper, C. A., N. Abramson, R. B. Johnson, J. H. Jandi, bacteremia and died. Thus, one complementand F. S. Rosen. 1970. Increased susceptibility to infection associated with abnormalities of complemediated protective effect in this model was the ment-mediated functions and of the third component limitation of the magnitude of bacteremia to a of complement (C3). N. Engl. J. Med. 282:349-354. sublethal level. This protective activity could 2. Alper, C. A., K. J. Bloch, and F. S. Rosen. 1973. Inthen allow time for other immune defense creased susceptibility to infection in a patient with type II essential hypercatabolism of C3. N. Engl. J. mechanisms, such as specific antibody syntheMed. 288:601-606. sis, to begin and thus result in the survival of 3. Alper, C. A., H. R. Colten, F. S. Rosen, A. R. Rabson,
the animal. In contrast to the effect on the incidence and magnitude of bacteremia, complement depletion did not directly influence the occurrence of meningitis. The magnitude of bacteremia at which the complement-depleted rats developed meningitis was the same as that in controls. CSF bacterial counts were also the same in both groups. Therefore, neither protection against CNS bacterial invasion nor control of bacterial multiplication within the CNS was a complement-related activity in this model. The lower CSF leukocyte counts in the CoVFtreated animals could reflect lowered chemotac-
4. 5.
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G. M. MacNab, and J. S. S. Gear. 1972. Homozygous deficiency of C3 in a patient with repeated infections. Lancet 2:1179-1181. Armitage, P. 1971. Statistical methods in medical research. John Wiley and Sons, New York. Easmon, C. S. F., and A. A. Glynn. 1976. Comparison of subcutaneous and intraperitoneal staphylococcal infections in normal and complement-deficient mice. Infect. Immunol. 13:399-406. Jacobs, J. C., and M. C. Miller. 1972. Fatal familial Leiner's disease: a deficiency of the opsonic activity of serum complement. Pediatrics 49:225-232. Johnson, R. B., M. R. Klemperer, C. A. Alper, and F. S. Rosen. 1969. The enhancement of bacterial phagocytosis by serum: the role of complement components and two cofactors. J. Exp. Med. 129:1275-1290. Miller, M. E., and U. R. Nilsson. 1970. A familial
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deficiency of the phagocytosis-enhancing activity of serum related to a dysfunction of the fifth component of complement (C5). N. Engl. J. Med. 282:354-358. Moxon, E. R., P. T. Ostrow, and R. T. Kapko. 1976. Experimental Hemophilus influenzae meningitis: relationship of bacteremia to CSF and histopathologic findings. Pediatr. Res. 10: 401. Moxon, E. R., A. L. Smith, D. R. Averill, and D. H. Smith. 1974. Haemophilus influenzae meningitis in infant rats after intranasal inoculation. J. Infect. Dis. 129:154-162. Newman, S. L., B. Waldo, and R. B. Johnston, Jr. 1973. Separation of serum bactericidal and opsonizing activities for Haemophilus influenzae, type b. Infect. Immun. 8:488-490. Ruddy, S., I. Gigli, and K. F. Austen. 1972. The complement system of man. N. Engl. J. Med. 287:289-495. Shin, H. S., H. Gewurz, and R. Synderman. 1969. Reaction of a cobra-venom factor with guinea-pig complement and generation of an activity chemotactic for polymorphonuclear leukocytes. Proc. Soc. Exp. Biol.
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Med. 131:203-207. 14. Shin, H. S., and M. M. Mayer. 1968. The third component of the guinea pig complement system. II. Biochemistry 7:2997-3002. 15. Shin, H. S., R. J. Pickering, and M. M. Mayer. 1971. The fifth component of the guinea-pig complement system. J. Immunol. 106:473-479. 16. Smith, D. H., D. L. Ingram, A. L. Smith, F. Gilles, and M. J. Bresnan. 1973. Bacterial meningitis: a symposium. Pediatrics 52:586-600. 17. Smith, D. H., G. Peter, D. L. Ingram, A. L. Harding, and P. Anderson. 1973. Responses of children immunized with the capsular polysaccharide of Hemophilus influenzae, type b. Pediatrics 52:637-644. 18. Winkelstein, J. A., H. S. Shin, and W. B. Wood. 1972. Heat labile opsonins to pneumococcus. III. The participation of immunoglobulin and of the alternative pathway of C3 activation. J. Immunol. 108:1681-1689. 19. Winkelstein, J. A., M. R. Smith, and H. S. Shin. 1975. The role of C3 as an opsonin in the early stages of infection. Proc. Soc. Exp. Biol. Med. 149:397-401.
