Oct 7, 1976 - according to the method of Kabat and Mayer. (8). Pooled normal guinea pig .... Charles C Thomas, Spring- field, Ill. 9. Lew, F. T., Y. Yasushi, ...
Vol. 16, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, Apr. 1977, p. 400-402 Copyright C 1977 American Society for Microbiology
Activation of the Alternative Complement Pathway by Haemophilus influenzae Type B PATRICIA HABER QUINN, FRANCIS J. CROSSON, JR., JERRY A. WINKELSTEIN,* AND E. RICHARD MOXON Departments ofPediatrics and Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Received for publication 7 October 1976
The results of the following study demonstrate that Haemophilus influenzae type B (HITB) is able to activate the alternative complement pathway. They also demonstrate that the activation of the alternative pathway by HITB is not mediated by the capsule.
The activation of complement components C3 through 9 (C3-9) plays a significant role in the host's defense against Haemophilus influenzae type B (HITB) (2). Two known pathways by which C3-9 can be activated exist, the classical pathway and the alternative pathway (11). It has been previously shown that a variety of both gram-negative and gram-positive bacteria are able to activate the alternative pathway (6, 13). However, studies to determine whether HITB is capable of activating the alternative pathway have produced conflicting results. In one study, HITB was able to activate C3 in C2deficient serum, suggesting that the organism might be capable of activating the alternative pathway (7). However, in another study, HITB was unable to activate complement in serum that had been chelated with ethyleneglycoltetraacetic acid, an inhibitor of the classical but not the alternative pathway. This suggested that the organism could not activate the alternative pathway (4). The following studies were performed to resolve the question of whether HITB is able to activate C3-9 via the alternative pathway. Veronal-buffered saline that contained gelatin, Ca2+, and Mg2+ (GVB2+) and GVB2+ that contained dextrose (DGVB2+) were prepared according to the method of Kabat and Mayer (8). Pooled normal guinea pig serum and pooled guinea pig serum obtained from animals with a genetically determined total deficiency of C4 (C4D GPS, 3) were each divided into small portions and stored at -70°C. An encapsulated strain of HITB (b-Eagan) and an untypable strain of H. influenzae completely devoid of any detectable capsule (U-I) were obtained from P. Anderson, The Harvard Medical School, Boston, Mass. (1), and cultured as previously described (10). The bacteria, in log-phase growth, were washed twice and suspended to the de-
sired concentration in GVB2 . Purified polyribosephosphate (PRP) was supplied by John B. Robbins, Bureau of Biologics, National Institutes of Health, Bethesda, Md. (5). Monospecific rabbit anti-guinea pig factor B was supplied by A. G. Osler, The Public Health Research Institute, New York, N.Y. (9). To determine whether HITB is able to activate C3 via the alternative pathway, the organism was suspended at a concentration of 6.25 x 108/ml in either normal guinea pig serum or in C4D GPS diluted 1:10 in GVB2+, and the mixtures were incubated at 37°C. The consumption of C3 was determined by obtaining samples at the desired interval, diluting them in icecold DGVB2+, and measuring the residual titer of C3 (12, 13). HITB was able to consume C3 in both C4D GPS and in normal guinea pig serum (Fig. 1). The observation that HITB was able to consume C3 in the absence of C4 suggested that HITB was able to activate the alternative pathway. To confirm that the consumption of C3 in C4D GPS was mediated by the alternative pathway, the conversion of factor B of the alternative pathway by HITB was examined (9). HITB was suspended at a concentration of 6.25 x 108/ml in C4D GPS diluted 1:2 in GVB2+ and incubated at 37°C for 30 min. Immunoelectrophoresis of the serum was then performed against rabbit anti-guinea pig factor B, and conversion of factor B from a beta to gamma migrating protein was observed, indicating activation of the alternative pathway (9). Thus, HITB is able to activate the alternative pathway. To determine whether the capsule contributes to the activation of the alternative pathway by HITB, the ability ofthe unencapsulated strain of untypable H. influenzae (U-I) to activate C3 in C4D GPS was compared to that of the encapsulated strain (b-Eagan). Both orga-
400
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VOL. 16, 1977
401
100 nisms were able to consume C3 (Fig. 2). In another experiment, purified capsular PRP *\ was suspended to the desired concentration in 90 C4D GPS that had been diluted 1:10 in GVB2, and the consumption of C3 was determined \ after a 30-min incubation at 370C. Concentra80 \ tions of PRP as high as 0.5 mg/ml failed to \ activate the alternative pathway (Fig. 3). Finally, since capsular PRP is attached to the '3 70 bacterial surface (1) when it exists in its na- .\ \ tive form, the ability of PRP to activate the . alternative pathway while attached to erythro- O 60 cytes, i.e., in a solid phase, was also examined. ' Sheep erythrocytes at a concentration of 109/ 50 o ml were incubated with PRP at a concentration of 1 mg/ml at 370C for 30 min; the PRP-coated 40 erythrocytes were then washed and could be agglutinated by burro anti-HITB antiserum. Such erythrocytes were incubated at a concen20 s 25 30 lo 15 tration of 109/ml in C4D GPS that had been Minutes diluted 1:10 in GVB2+ for 30 min at 370C and did not consume C3 when compared to control FIG. 2. C3 consumption by encapsulated (@) and erythrocytes. Thus, the HITB capsule does not unencapsulated (0) H. influenzae in C4D GPS. appear to activate the alternative pathway. As with other gram-negative organisms, the ac100 tivation of the alternative pathway by HITB is probably mediated by an endotoxic lipopoly90 saccharide of the cell wall. The activation of C3-9 plays a significant role v 80 in the defense of the host against HITB infec- E 70 tion (2). Previous studies have shown that " 7 HITB, when complexed with antibody, will ac- 8 60 tivate C3-9 via the classical pathway (1). The to present studies directly demonstrate that HITB Q 50 can also activate C3-9 via the alternative path- ° 40 way. Whether HITB will activate the classical c4 -
-
-
-
30
V
100
-
20
90 -~~~~~~~~~~
20
100
200
Polyribosephosphate
400
500
(,1g/ml)
FIG. 3. C3 consumption by PRP in C4D GPS.
O 80 C
so\ \ _ 70
*
* a.
60 -0 500
0
5
0o
15s
20,
25,
B
30
Minutes FIG. 1. C3 consumption by HITB in normal guinea pig serum (*) and in C4D GPS (0). C7
--7--
----,-,----
upon depends or alternative of antibody absence probably and, thus, the presence orpathway the immune status of the host. Presumably, activation of C3-9 via the classical pathway plays a role in acquired immunity to HITB, whereas activation of C3-9 via the alternative pathway is important in natural immunity to HITB. This work was supported by Public Health Service grants AI-11637 and AI-00461 from the National Institute of
Allergy and Infectious Diseases, by Public Health Service
grant NS-12554 from the National Institute of Neurological
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NOTES
and Communicative Disorders- and Stroke, and by the Hospital for Consumptives of Maryland (Eudowood). Patricia Haber Quinn was a Research Associate of the Howard Hughes Medical Institute. Jerry A. Winkelstein is an Investigatar of the Howard Hughes Medical Institute. LITERATURE CITED 1. Anderson, P., R. B. Johnston, Jr., and D. H. Smith. 1972. Human serum activities against Hemophilus influenzae, type B. J. Clin. Invest. 51:31-38. 2. Crosson, F. J., Jr., J. A. Winkelstein, and E. R. Moxon. 1976. Participation of complement in the nonimmune host defense against experimental Haemophilus influenzae type b septicemia and meningitis. Infect. Immun. 14:882-887. 3. Eliman, L., I. Green, and M. Frank. 1970. Genetically controlled complete deficiency of the fourth component of complement in the guinea pig. Science 170:7477. 4. Fine, D. P., S. R. Marney, Jr., D. G. Colley, and R. M. Des Prez. 1973. Hemophilus influenzae decomplementation pattern in chelated and nonchelated serum, p 113-117. In S. H. W. Sell and D. T. Karzon (ed.), Hemophilus influenzae: Proceedings of a Conference on Antigen-Antibody Systems, Epidemiology, and Immuno-Prophylaxis. Vanderbilt University Press, Nashville, Tenn. 5. Gotschlich, E. C., T. Y. Liu, and M. S. Artenstein. 1969. Human immunity to the meningococcus. II. Preparation and immunochemical properties of the group A, group B and group C meningococcus polysaccharides. J. Exp. Med. 129:1349.
INFECT. IMMUN. 6. Gotze, O., and H. J. Muller-Eberhard. 1971. The C3activator system: an alternative pathway of complement activation. J. Exp. Med. 134:905-975. 7. Johnston, R. B., Jr., P. Anderson, and S. L. Newman. 1973. Opsonization and phagocytosis of Hemophulus influenzae, type B, p. 99-112. In S. H. W. Sell and D. T. Karzon (ed.), Hemophilus influenzae: Proceedings of a Conference on Antigen-Antibody Systems, Epidemiology, and Immuno-Prophylaxis. Vanderbilt University Press, Nashville, Tenn. 8. Kabat, E., and M. M. Mayer. 1961. Experimental immunochemistry, 2nd ed. Charles C Thomas, Springfield, Ill. 9. Lew, F. T., Y. Yasushi, H. S. Waks, and A. G. Osler. 1975. Activation of the alternative (properdin) pathway by divalent cations. J. Immunol. 115:884-888. 10. Moxon, E. R., A. L. Smith, D. R. Averill, and D. H. Smith. 1974. Hemophilus influenzae meningitis in infant rats after intranasal innoculation. J. Infect. Dis. 129:154-162. 11. Ruddy, S., I. Gigli, and K. F. Austen. 1972. The complement system of man. Activation, control and products of the reaction sequence. New Engl. J. Med. 287:489495. 12. Shin, H. S., and M. M. Mayer. 1968. The third component of the guinea pig complement system. Biochemistry 7:2997-3002. 13. Winkelstein, J. A., H. S. Shin, and W. B. Wood, Jr. 1972. Heat labile opsonins to pneumococcus. III. The participation of immunoglobulin and of the alternative pathway of C3 activation. J. Immunol. 108:16811689.
