Neisseria meningitidis

Transcription

1 INFECTION AND IMMUNITY, June 1971, p Copyright 1971 American Society for Microbiology Vol. 3, No. Printed ill U.S.A. Cellular Response of the Rabbit Eye to Primary Intravitreal Inj ection of Neisseria meningitidis JAMES F. PRIBNOW, JOAN M. HALL,' DIANE BRADLEY, AND NEYLAN A. VEDROS Naval Biomedical Research Laboratory, School of Public Health, University of Californlia, Berkeley, California Received for publication 4 March 1971 New Zealand White rabbits were injected intravitreally with approximately 2,500 viable or heat-killed meningococci. The rabbits were killed at intervals from 30 min to 14 days after injection. None of the rabbits produced detectable antibodies. Local antibody production by uveal tract, spleen, or preauricular lymph node was not demonstrated. Viable organisms were recovered from the vitreous at periods from 30 min to 48 hr after injection. Failure to recover viable organisms could be correlated with the appearance of large numbers of polymorphonuclear neutrophiles (PMN) throughout the vitreous. Animals injected with viable meningococci demonstrated a progressive inflammatory reaction characterized by an early accumulation of PMN in the vitreous, limbus, and conjunctiva. The cellular infiltrate gradually became. By the 14th day postinjection only a few residual inflammatory remained at the limbus. This extensive cellular response was lacking in recipients of heat-killed organisms. The defense of the rabbit against intraocular introduction of meningococci therefore seems to be predominantly a cellular mechanism. Information regarding the role of cellular immunity in meningococcal infection is important for a fuller understanding of the disease (1). Humoral bactericidal activity (4) and opsonins (12) have been implicated as major determinants of resistance. However, animals such as mice, which lack these humorally mediated attributes, are refractory to meningococcal disease. The possible role of a cellular component of resistance to Neisseria meningitidis was therefore investigated. Our use of the ocular route of injection was prompted by several studies. Intravitreal injection of protein antigens has been shown to be an effective method of inducing antibody formation in rabbits (5, 11, 14). A hemolytic plaque technique has been adapted to detect uveal tract producing antibody to bovine gamma globulin () and to sheep cell stroma (15). Miller et al. (10) produced antibody to the gonococcus after injection of viable organisms into the anterior chamber of the rabbit eye. Recently, the plaque technique has been adapted by Evans (3) for use with a N. meningitidis antigen. However, the number of in the mouse spleen which produced antibody specific for the menin- I Present address: Francis 1. Proctor Foundation for Research in Ophthalmology, University of California, San Francisco, Calif. 739 gitidis antigen was low. Our supposition was that injection of meningococci into the vitreous might result in the production of humoral antibody and that significant numbers of plaque-forming might be detected in the uveal tract tissue. We also investigated the possibility of whether ocular tissues would support the growth of the intravitreally injected meningococci. Our findings showed that the vitreous or associated ocular tissues, or both, supported survival and limited multiplication of the organisms injected, even though overt clinical infection was not apparent. Antibody was not detected in serum or vitreous. However, a self-limited inflammatory reaction did ensue. These results, which differ from those obtained after intravitreal injection of other organisms, suggest that a cellular defense mechanism may play a vital role in host resistance after introduction of meningococci. MATERIALS AND METHODS Microorganisms. N. meniingitidis (group A, strain 791), isolated from the spinal fluid of a patient with spinal meningitis and maintained within three passages from the source, was used exclusively in this study. Seed cultures were maintained at -70 C. The bacteria were cultivated on Mueller Hinton (Difco)

2 740 PRIBNOW ET AL. INFEC. IMMUN. agar slants for 18 hr at 37 C (10% CO2). The were washed once in Hanks balanced salt solution (HBSS) and resuspended in HBSS at a cell density adjusted spectrophotometrically to 108 per ml. The heat-killed were inactivated by heating for 30 min at 0 C. Experimental animals. New Zealand White rabbits, weighing approximately 2.5 kg, were used in this study. Injection procedure. A topical anesthetic (0.5% proparacaine hydrochloride ophthalmic solution, E. R. Squibb & Sons, New York, N.Y.) was applied to the surface of the eye immediately before intravitreal injection. The eye was proptosed, and 0.05 ml of viable or heat-killed bacteria (approximately 2.5 X 103 ) was injected into the vitreous by using a tuberculin syringe and a 27-gauge needle. Viability of the inoculum was determined by culturing a sample of each suspension immediately before injection. The possibility that vitreous might be bactericidal was investigated by using both in vitro and in vivo methods. Samples of normal vitreous fluid removed aseptically from enucleated eyes were inoculated with viable organisms. Samples were plated at 0, 3.5, and 24 hr. For the in vivo studies, portions of the aspirated vitreous from eyes enucleated at periods from 30 min to 72 hr were plated on Mueller Hinton medium. Serial dilutions were performed to ascertain the number of organisms recovered. Antibody determinations. Serum samples were obtained by bleeding the rabbits from the marginal ear vein at intervals after injection. Vitreous samples were obtained at the time the rabbits were killed. All samples were tested by a standard bacterial agglutination procedure and also by passive hemagglutination. In the latter test, the erythrocytes were coated with a somatic antigen prepared by the method of Sanborn and Vedros (12). Controls consisted of known positive sera, normal sera, and uncoated erythrocytes. Plaque assay. The enucleated eyes of rabbits killed at various times after intravitreal injection were kept at 4 C until used (10 min or less). The vitreous fluid was aspirated, and samples were plated on Mueller Hinton agar. Smears were prepared for Giemsa staining, and the remainder of the material was used for antibody determination and in the plaque assay. The anterior uvea was stripped from the underlying sclera, and a cell suspension was prepared in medium 199 (Microbiological Associates, Albany, Calif.). The were washed with the same medium, and the plaque assay was carried out as described by Hall and O'Connor (). Plaque assays were also done on spleen and preauricular lymph node cell suspensions. Erythrocytes were coated with somatic antigen by using a concentration of 20 jug of protein per 0.1 ml of (3). Histological procedures. Formalin-fixed uveal tract tissue and, in some cases, entire eyes were processed by standard histological techniques and stained with hemotoxylin and eosin and with methyl green pyronin (MGP) stains. Some specimens were also stained by the MacCallum-Goodpasture procedure. RESULTS Clinical observations. Eyes injected with viable N. meningitidis remained grossly normal for the first 2 days after injection. Most eyes then developed moderate to severe uveitis, characterized by conjunctivitis and iritis. This inflammation persisted until the th to 7th day after injection and gradually subsided. Many eyes appeared clinically normal by the 9th day postinjection, and no evidence of inflammation remained by the 14th day. Figure 1 shows the appearance of the eye 4 days after injection of viable meningococci. Rabbits injected with a similar number of heat-killed bacteria did not develop this inflammatory reaction. Occasionally a slight iritis and conjunctivitis, possibly due to the trauma of injection, were observed. This reaction subsided within a few hours. Bacteriological studies. Aspirated vitreous was not bactericidal for organisms cultured for 3.5 or for 24 hr. Viable organisms were recovered from the vitreous at periods of from 30 min to 48 hr after injection of viable N. meningitidis. Organisms recovered were gram-negative diplococci and were oxidase-positive. No growth occurred in cultures of vitreous removed 72 hr after injection. Table 1 gives the results of experiments in which the number of organisms recovered at various times after a single injection of meningococci was determined. Although these results can only be considered semiquantitative because a small amount of the inoculum often leaks from the vitreous, there is clear indication of at least limited growth of the organisms for the first 48 hr after injection. Growth of organisms was also noted in cultures established 18 and 3 hr after injection when 1.5 X 103 organisms were injected. FIG. 1. External appearance of the rabbit eye 4 days after injection of viable Neisseria meningitidis, showing marked conjunctivitis and iritis.

3 VOL. 3, 1971 RESPONSE OF RABBIT EYE TO N. MENINGITIDIS 741 Plaque assay. Cells from the uveal tracts, vitreous, preauricular lymph nodes, and spleens of rabbits killed at periods of from 2 to 14 days after injection were assayed for antibody production. No plaques were produced by uveal tract or in the vitreous. Plaques produced by lymph node or spleen did not exceed the background count for noncoated erythrocytes. Therefore, we concluded that the tissues examined did not contain which produced specific antimeningococcal antibody. Antibody determination. Antibodies to somatic antigen or to viable N. meningitidis were not detected in the serum of the rabbits. The vitreous samples also failed to show antibody to these antigens. Examination of vitreous smears. Giemsa stains were performed on smears of the vitreous aspirated after enucleation. The results of these examinations are presented in Table 2. The progression from a predominantly polymorphonuclear reaction to a reaction is evident. The identity of organisms morphologically resembling Neisseria was confirmed by the Gram stains. Both free organisms and intracellular organisms were observed at early periods. Examination of tissue sections. The histological response to injection of viable organisms is TABLE 2. Examinationi of aspirated vitreous fluid Time after injection 30 min, 1, 2, 4, 8 hr 12 hr 18 hr 24 hr 3 hr 48 hr 72 hr 4 days days 9 days 11 days 14 days No. of eyes injected Six for each time presented in Table 3. Some variation in the severity of the inflammatory response was noted. Rabbits that had received fewer organisms generally responded less vigorously. Ocular tissues other than those described in the table were minimally involved. Blood vessels were seen along the retina, and some polymorphonuclear TABLE 1. Recovery of organisms from vitreous fluid after injection of viable Neisseria meningitidis Time (hr) No. of No. of organisms No. of organisms after organisms recovered per ml recovered per injection injected of vitreous eye" 30b 9.5 X X 10'c.87 X 103 0b 9*5 X X X X X X X X X X X X X X X X X X X X a Approximate number of organisms recovered from vitreous based on an average vitreous volume of 3.5 ml. bminutes. c Average count from four eyes. after intravitreal injection of viable meningococci No. of organisms injected 9.5 X X X X X X 103 to 2.4 X X X 102 to 2.1 X X 102 to 2.8 X X 102 to 3.7 X X 102 to 2. X X 102 to 2. X 103 a Polymorphonuclear neutrophiles. b Term macrophages is used to designate large phagocytic. Results Gram-negative diplococci Gram-negative diplococci; widely scattered PMNa; rare Many gram-negative diplococci; scattered PMN Gram-negative diplococci; moderate number of PMN, some containing diplococci; rare Gram-negative diplococci; many PMN, some containing diplococci; rare Many PMN, some containing organisms; gram-negative diplococci; scattered macrophages Many PMN, few macrophages; some gram-negative diplococci Many PMN, a few macrophages Macrophages containing organisms or intact PMN, or both; a few PMN Macrophages, a few PMN A very few macrophages No intact

4 742 PRIBNOW ET AL. INFEC. IMMUN. TABLE 3. Histology of the inflammatory response after intravitreal inijection Time after injection Conjunctix-a Limbus Ciliary Ciliary body of viable meninzgococci Anterior ~vitreous' peripheral 30 min to 1 hr 2, 4, 8, and 12 hr 18 and 24 hr a 3 hr 2 days 3 and 4 days days 9 days 11 days 14 days A few PMN5 and c A few to moderate numbers of PMN; some Moderate number PMN; some infiltrate; predominantly PMN, some Moderate number of PMN and number PMN; Mononuclear ; fewer PMN A few Essentially normal A very few PMN and PMN, fewer than in conjunctiva; some Moderate number of PMN; some PMN and PMN infiltrate; some Extensive infiltrate; PMN and infiltrate; PMN and Mild to moderate infiltrate; predominantly Rare PMN and PMN in some sections PMN in some sections Moderate number of PMN Moderate infiltrate; predominantly PMN PMN and Mononuclear Rare PMN and Essentially normal A few PMN and exudate; predominantly PMN Extensive exudate; predominantly PMN Exudate, about equal numbers of PMN and macrophages A few macrophages Essentially normal This term is used to describe the portion of the vitreous overlying the ciliary body and ciliary processes. b Polymorphonuclear neutrophiles. c Mononuclear refer to all such observed. The term macrophage is reserved for large phagocytic. neutrophiles (PMN) were observed in the posterior vitreous during the first 2 days of the reaction. Corneal involvement appeared to be limited to PMN invasion of the corneal stroma adjacent to the limbus and was noted at 4 and days only in those eyes which showed the most intense reaction. Figures 2, 3, and 4 show the histological appearance of the ocular tissue at various times after injection of viable organisms. The histological response to heat-killed organisms differed strikingly from that described in Table 3. The most prominent feature was the appearance of a few and rare PMN at the limbus, conjunctiva, and ciliary body. The response to heat-killed organisms was essentially the same at all time periods. At no time was the extensive infiltrate into the anterior peripheral portion of the vitreous observed. Figure 5 shows the limbus of one of these rabbits. The MGP-stained sections revealed only scattered pyroninophilic at the limbus, conjunctiva, and ciliary body. The extensive infiltration of the anterior uvea with pyroninophilic which is seen in the response to protein antigens was lacking. The MacCallum-Goodpasture stain was performed on sections of eyes enucleated the first 4 days after injection. Gram-negative diplococci were seen in the vitreous of all sections examined. Organisms were occasionally seen in the anterior

5 ax a),2 VOL. 3, 1971 RESPONSE OF RABBIT EYE TO N. MENINGITIDIS SS r* V.;.;, ( 74. * t * c } * v fl >*t. a Sx z t3f pt t g1 irb *~d : wt 5 X < t ' S * & r W s^tvx e~~~~~~~~~~~~~~~~~~~~~~. ~ ~ ~ ~ ~ X g 4 W7&; S 4 t& ; i s FIG. 2. Anterior peripheral vitreous 4 days postinjection of viable Neisseria meningitidis, sh7owing extensive polymorphonuclear exudate. X e. :::,. :-. FIG. 3. Limbus and conjunctiva days after injection of viable Neisseria meningitidis, showing extensive infiltrate of inflammatory. Areas ofcornea and sclera are also affected. X 78. chamber. Because of the highly granular nature of rabbit neutrophiles, it was not possible to state that PMN infiltrating the limbus and conjunctiva contained phagocytized organisms. Organisms were seen in aspirated from the vitreous. Many of the phagocytic seen in the anterior vitreous contained organisms. Some of these macrophage-like also contained intact PMN. DISCUSSION Drell et al. (2) observed that injection of gonococci into the anterior chamber of the rabbit eye resulted in the production of complementfixing antibodies. On rare occasions, small amounts of agglutinating antibody were produced. In our study, injection of 2 X 103 meningococci into rabbits' eyes did not result in the production of serum or vitreous antibodies detectable by passive hemagglutination or bacterial agglutination. None of the tested in the plaque assay produced antibodies capable of lysing sheep erythrocytes coated with somatic antigen. The efficacy of the coating procedure was tested by complement-dependent lysis of coated

6 744 PRIBNOW ET AL. INFEC. IMMUN. A7:.::4 if~~~~ A PO~~~a awō ~~ /,d%4/ 4.4,/~No FIG. 4. Limbus 9 days after injection of viable Neisseria meningitidis, showing predominantly infiltrate. X 200. :.:.... :.; }. FIG. 5. Limbus and conjunctiva days after injection of heat-killed Neisseria meningitidis, showing absence of cellular infiltrate. Contrast with Fig. 3 is striking. X 78. contained in agarose, using a known positive serum. The lack of antibody production was to some extent confirmed by examination of the MGP-stained sections. Only rare pyroninophilic were seen in the anterior uvea, the majority occurring at the limbus and conjunctiva. These tissues are technically difficult to assay for antibody-producing. In contrast to our results, intravitreal injection of some other microorganisms has resulted in antibody production. Witmer (17) was able to demonstrate agglutinating antibodies to Leptospira, and Wolkowicz et al. (18) was successful in detecting antibody after injection of typhoid bacilli. The vitreous fluid has been shown to support the growth of several microorganisms. Maylath and Leopold (8, 9) showed that intravitreal injection of as few as 700 S. aureus, P. aeruginosa, or E. coli could initiate an infection. The organisms were isolated from vitreous, retina, and anterior chamber as late as 72 hr after injection. Witmer (17) cultured L. pomona for 1 week after intravitreal injection of 2 X 10 organisms. Attempts to culture Neisseria in ocular tissue CO

7 VOL. 3, 1971 RESPONSE OF RABBIT EYE TO N. MENINGITIDIS 745 have not been as successful. Miller et al. (10) injected gonococci into the anterior chamber of rabbit eyes. Organisms were cultured from aqueous, from ciliary body, and from the lens surface. When 2,000 organisms, approximately the number used in the majority of our studies, were injected, only 4% of the eyes became infected. When 2 X 107 organisms were used, organisms were recovered from 95 % of the eyes. The authors believe that the organisms multiplied in the ocular tissue and did not merely remain viable. We assume that had the vitreous been meningococcidal, we would not have observed the vastly different cellular reaction obtained after injection of heat-killed or viable organisms. Vitreous fluid was not bactericidal either in vitro or in vivo. Our evidence indicated that the vitreous actually supported the growth of the meningococci. More organisms were recovered at 18, 24, 3, and 48 hr than had been present in the original inoculum. This was true even when as few as 1.5 X 103 organisms were injected. The apparent decrease in the number of organisms recovered for the first few hours may be due to the fact that organisms had not dispersed far from the site of injection. The needle was probably introduced into a different site for aspiration. Fewer organisms were also seen in the stained preparations at these early times. The number of organisms recovered from the eyes may be minimal, as it does not take into account organisms possibly present in the anterior chamber or ocular tissues or those phagocytized by inflammatory. No organisms were recovered after 72 hr, even though bacteria morphologically resembling Neisseria were sometimes seen in the aspirated vitreous. These organisms were apparently no longer viable. Phagocytosis of the injected organisms probably accounts for the failure to culture them later than 72 hr. There appears to be a definite correlation between recovery of viable organisms and the appearance of an inflammatory exudate in the region of the vitreous overlying the iris and ciliary body. Also, at this time the infiltrate into the limbus and conjunctiva was much more extensive and the rabbits developed clinical uveitis. Many of the in the anterior vitreous appeared actively phagocytic, especially those macrophages which contained phagocytized PMN. Perhaps the most interesting aspect of this study was the cellular reaction which occurred after intravitreal injection of viable organisms. This reaction was very different temporally and histologically from that which occurs after injection of a soluble protein antigen or a particulate antigen such as sheep erythrocytes. The eyes of rabbits injected with these latter antigens remain normal until the development of uveitis on about the 7th day after injection. Our rabbits injected with viable meningococci developed uveitis by the 2nd or 3rd day postinjection. The primary cellular reaction to protein antigens is, beginning at the onset of uveitis and persisting several days. An extensive infiltrate of pyroninophilic is observed in the ciliary body, iris, and ciliary processes. In contrast, the initial response to meningococci is polymorphonuclear and is confined mostly to the vitreous, limbus, and adjacent conjunctiva. Histologically the cellular response was similar to that observed by Drell et al. (2) after injection of gonococci into the anterior chamber of rabbit eyes. The reaction is also very similar to that noted by Larson (7), who injected viable N. meningitidis in subcutaneous enclaves in mice. The progression from PMN to occurred more rapidly, perhaps because of the greater accessibility of the injection site to the circulation. In addition, a very early reaction was noted which lasted from immediately after injection until 2 hr postinjection. The reaction to heat-killed organisms was very different from that described above and probably represented the normal reaction to foreign material in the vitreous. It might be argued that heat-killed organisms are not the best control for viable organisms. However, it has been shown that heat killing of meningococci is an effective way of preventing autolysis and alteration of surface antigenic components (1) İt seems apparent that the cellular defense mechanisms of the rabbit were able to cope adequately with the intravitreal introduction of at least 2,500 viable organisms, as only limited multiplication of the organisms took place and the cellular reaction was self-limited. The contrast between the reaction to heat-killed and viable organisms indicated that viable are necessary to bring about the cellular reaction observed. The factor or factors in viable which account for this difference are presently unknown. The results of these investigations with N. meningitidis indicate that intraocular injection may be an effective tool for studying the cellular events which occur in the defense against certain microorganisms. The response to challenge injection is currently under investigation. ACKNOWLEDGMENTS The authors express their appreciation to Charles S. Wilkey of the Proctor Foundation for his valuable technical assistance and to G. Richard O'Connor for his review of the manuscript. This investigation was supported by the Office of Naval Re-

8 74 PRIBNOW ET AL. INFEC. IMMUN. search under a contract between the Office of Naval Research and the Regents of the University of California and grant no. EY00310 from the National Institutes of Health, Bethesda, Md. LITERATURE CITED 1. Branham, S. E., H. E. Alexander, C. R. Falk, M. H. Lepper, and L. Weinstein Bacterial meningitis, p In A. H. Harris and M. B. Coleman (ed.), Diagnostic procedures and reagents. American Public Health Association, Inc., New York. 2. Drell, M. J., C. P. Miller, M. Bohnhoff, and V. Moeller Experimental gonococcal infection of the rabbit's eye. II. Course of the disease and its pathology. J. Infec. Dis. 77: Evans, D. G Detection of antibody-producing of mice injected with antigens of Neisseria meniingifidis. Infec. Imnmun. 1: Goldschneider, I., E. C. Gotschlich, and M. S. Artenstein Human immunity to the meningococcus. II. Development of natural immunity. J. Exp. Med. 129: Hall, J. M., and G. R. O'Connor Correlation between ocular inflammation and antibody production. I. Serum antibody response following intravitreal immunization with protein antigens. J. Immunol. 104: Hall, J. M., and G. R. O'Connor Correlation between ocular inflammation and antibody production. HI. Hemolytic plaque formation by of the uveal tract. J. Immunol. 104: Larson, A Subcutaneous enclaves in mice as sites for the study of cellular responses inl vivo. J. Bacteriol. 97: Maylath, F. R., and I. H. Leopold Study of experimental intraocular infection. I. The recoverability of organisms inoculated into ocular tissues and fluids. Amer. J. Ophthalmol. 40: Maylath, F. R., and I. H. Leopold Study of experimental intraocular infection. II. The influence of antibiotics and cortisone, alone and combined, on intraocular growth of these organisms. Amer. J. Opthalmol. 40: Miller, C. P., M. J. Drell, V. Moeller, and M. Bohnhoff Experimental gonococcal infection of the rabbit's eye. I. Method of production. J. Infec. Dis. 77: Park, J. J., H. M. Leibowitz, and A. E. Maumenee The effect of route of inoculation upon development of antibody in rabbits. J. Immunol. 87: Roberts, R. B The relationship between group A and group C meningococcal polysaccharides and serum opsonins in man. J. Exp. Med. 131: Sanborn, W. R., and N. A. Vedros. 19. Possibilities of application of complement fixation, indirect hemagglutination and fluorescetit antibody tests to epidemiology of meningococcal infection. Health Lab. Sci. 3: Silverstein, A. M Ectopic antibody formation in the eye: pathologic implications, p In A. E. Maumenee and A. M. Silverstein (ed.), Immunopathology of uveitis. Williams and Wilkins Co., Baltimore. 15. Smith, R. E., A. D. Jensen, and A. M. Silverstein Antibody formation by single during experimental immunogenic uveitis. Invest. Ophthalmol. 8: Vedros, N. A., D. H. Hunter, and J. H. Rust. 19. Studies on immunity in meningococcal meningitis. Mil. Med. 131: Witmer, R. H Experimental leptospiral uveitis in rabbits. Arch. Ophthalmol. 53: Wolkowicz, M. I., J. W. Hallet, and I. H. Leopold Studies on antibody production in experimental uveitis. I. Antibody formation in vitro after intraocular injection of typhoid bacilli into the rabbit eye. Doc. Ophthalmol. 14:50-3. Downloaded from on June 30, 2018 by guest