Vol. 22, No. 3

INFECTION AND IMMUNITY, Dec. 1978, p. 926-933 0019-9567/78/0022-0926$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Heightened Resistance of Athymic, Nude (nu/nu) Mice to Experimental Pseudomonas aeruginosa Ocular Infection L. D. HAZLETT`*

AND

R. S. BERK2

Departments of Anatomy' and Immunology/Microbiology,2 Wayne State University, Detroit, Michigan 48201 Received for publication 4 October 1978

BALB/c-derived athymic, nude (nu/nu) mice exhibited a heightened natural resistance to experimental corneal infection with Pseudomonas aeruginosa when compared with their heterozygote (nu/+) littermates. Stereomicroscopic examination of the eyes of nu/nu mice 24 h after corneal trauma and topical bacterial application revealed slightly cloudy corneas (iris visible), whereas nu/+ littermate corneas were opaque (iris not visible). Nu/+ mice failed to resolve the infection, and endophthalmitis and shrinkage of the infected eye occurred in these mice within 2 weeks after experimental Pseudomonas infection, as in the parent BALB/c strain. However, nu/nu mice, similarly infected, resolved the infection within 24 h and never exhibited full corneal opacity or eye shrinkage. Histological examination of the corneas of nu/nu mice 24 h after experimental wounding and bacterial application demonstrated subepithelial capillaries and a few polymorphonuclear neutrophils (with numerous intracellular bacteria) in the central cornea. In contrast, the equivalent corneal areas of infected nu/+ littermates, examined similarly, showed a more striking neutrophilic response (but with few intracellular bacteria) to similar bacterial infection, as well as a lack of blood vessels within the central cornea. The central corneas of uninfected and saline control nu/nu mice also were observed. This area in nu/nu mice exhibited an infrequent polymorphonuclear neutrophil (with no intracellular bacteria) and capillaries similar in size and location to those described for experimentally infected nu/nu mouse corneas. Untreated and saline control nu/+ mice, on the other hand, lacked both vessels and polymorphonuclear neutrophils in the central cornea.

Previous studies in our laboratories with naturally resistant Swiss Webster and susceptible BALB/c mice not carrying the nude gene indicated that both strains of mice initially respond to experimental Pse4domonas corneal infection with a marked accumulation of polymorphonuclear neutrophils at the infection site. These cells never are observed within the corneas of either mouse strain prior to infection. The Swiss Webster strain spontaneously resolves the infection, whereas the BALB/c strain undergoes endophthalmitis with subsequent eye shrinkage (phthisis bulbi) (3). The purpose of the present study is to determine if thymic deficiency has an effect on the course of experimental Pseudomonas ocular infection. Therefore, congenitally athymic, nude (nu/nu) mice were chosen as a model for this study since they are animals which, because of their genetic constitution, are immunoincompetent. These mice lack mature T-cells due to an embryological failure of thymic development (7), and therefore immune responses to thymus dependent antigens are either

absent or reduced (6). Nevertheless, recent evidence has shown that nu/nu mice possess a heightened resistance to experimental systemic infection with several bacterial agents (1, 2, 5, 8), despite the fact that immune protection against these pathogens requires a cell-mediated immune response. To account for this increased resistance to bacterial infection in nu/nu mice, it has been suggested that their macrophages possess enhanced microbicidal activities (1, 5, 8). This effect recently was demonstrated to be a direct consequence of the thymic deficiency (5). The present study compares and contrasts the in vivo response to Pseudomonas ocular infection and contributes morphological observations on BALB/c-derived nu/nu mice and their nu/+ littermates. The latter observations may, in part, contribute to the unexpected heightened resistance of nu/nu animals to experimental pseudomonas ocular infection, as compared to the previously described susceptible parent BALB/c strain and the naturally resistant Swiss Webster mouse strain (3), as well as nu/+ littermates. 926

RESISTANCE OF nu/nu MICE TO P. AERUGINOSA 927 MATERIALS AND METHODS sera from both the nu/nu and heterozygote littermates

VOL. 22, 1978

Stock cultures of P. aeruginosa strain 19660 ATCC stored at 250C on tryptose agar slants (Difco, Detroit, Mich.) were used for inoculation of 50 ml of broth medium containing 5% peptone (Difco) and 0.25% Trypticase soy broth (Baltimore Biological Laboratories, Cockeysville, Md.). The culture was hemolytic and proteolytic and produced lecithinase and exotoxin A. The culture was grown on a rotary shaker at 370C for 18 h, centrifuged at 27,000 x g for 20 min (40C) and suspended in 0.9% sterile saline (Travenol Laboratories Inc., Deerfield, Ill.) to the desired concentration of 5.0 X 1010 colony-forming units (CFU)/ml by using a predetermined curve relating viable counts to optical density at 440 nm. To establish the number of viable cells, serial dilutions were plated onto tryptose agar plates and read for growth at 24 h. BALB/c-derived athymic, nude (nu/nu) mice (69 mice) and their heterozygote (nu/+) littermates (52 mice) 1 to 2 months of age were obtained via Kenneth Morrison from a colony maintained at the Department of Immunology/Microbiology, Wayne State University School of Medicine. The colony was established in 1974 from heterozygotes of a ninth generation BALB/c backcross, generously supplied by Donald Shreffler of Washington University. The room in which the colony was housed contained a horizontal laminar air filtration unit (Environmental Air Control, Inc., Hagerstown, Md.) which nimized the number of microbes within the immediate environment. Drinking water containing Chlorox at a concentration of 200 ppm was provided ad libitum. Except for these strict precautions and sterilization of feed, bedding and cages, the mice were maintained in a conventional manner. In each of three separate experiments, using similar numbers of mice, as indicated above, animals were lightly anesthetized with ether and placed beneath a stereomicroscope, and the corneal surface was incised three times with a sterile 26-gauge needle, taking care not to penetrate into the anterior chamber (3). A transient, minimal cellular response normally occurs in the heterozygote after corneal abrasion but disappears within 24 h. Little or no response is observed in nude mice. Final concentrations of bacterial cell suspensions (1.0 X 107 to 1.0 x 108 CFU) were delivered topically onto wounded corneas. Control mice received sterile saline topically onto wounded corneas as described above. Randomly selected untreated mice also were observed. Both eyes of all animals were examined daily with a stereomicroscope at X40. Mice were sacrificed by ether overdose either prior to, or at 24 h after, bacterial challenge for fine structural analysis. The eyes were enucleated, fixed whole in a mixed fixative (1% osmium tetroxide and 2.5% glutaraldehyde) in Sorenson's phosphate buffer (pH 7.4 at 4.0°C for 3 h), dehydrated in graded ethanols, and embedded in Epon-Araldite. Thick sections (1.5 pm) were cut, stained with 1% toluidine blue, observed, and used to orient the central cornea for thin sectioning. Silver thin sections were cut, stained with uranyl acetate and lead citrate and observed and photographed with a Zeiss EM 9S-2 electron microscope. In addition, no agglutinin titers specific for P.

aeruginosa were detected in separately pooled mouse

prior to ocular infection.

RESULTS

Stereomicroscopic examination of the eyes of nu/nu mice 18 to 24 h through 21 days after infection with 107 to 108 CFU of Pseudomonas per ml reveals slightly cloudy to clear corneas in

all of the experimentally infected nu/nu mice. At least 60% of all nu/nu mice examined before, and at 24 h after, experimental Pseudomonas infection demonstrated blood vessels in the central cornea which appeared to be fine branches of adjacent limbal capillaries. All nu/nu mice

however, responded similarly to experimental Pseudomonas infection whether or not blood vessels were detected upon stereomicroscopic examination. No significant differences between nu/nu saline control corneas and bacterially infected corneas were observable, nor did any of the infected nu/nu mice exhibit eye shrinkage throughout 21 days of observation. On the other hand, examination of nu/+ littermate corneas 18 to 24 h after similar bacterial infection, revealed corneal opacity, with the iris not visible, in mice receiving 107 to 10i CFU of Pseudomonas per ml. The infection always remained localized, not spreading to the uninfected eye, to other body tissues, or to other mice. Within 12 to 15 days, nu/+ mice exhibited shrunken eyes, similar to the degenerative changes previously reported for the parent BALB/c mouse strain not carrying the nude gene (3). No blood capillaries were observed in the central corneas of any nu/+ mice before, or at 24 h after, experimental bacterial infection. The central corneas of nif/nu mice were examined ultrastructurally and with light microscopy before and at 24 h after either bacterial infection or topical saline application. In both the untreated (Fig. 1) and the experimentally infected (Fig. 2) nu/nu corneas examined in this manner, capillaries of varying caliber were found beneath the epithelial surface; however, bacteria were observed only in experimentally infected nu/nu corneas (Fig. 2 and 2a). The walls of the capillaries were composed of one or more endo-

thelial cells which were either attenuated and fenestrated (Fig. 3) or thickened and non-fenestrated (Fig. 4). Both fenestrated and nonfenestrated capillaries were surrounded by a thin basal lamina and an occasional pericyte (Fig. 3). Local basal lamina thickenings and platelet accumulation were seen only in capillaries with attenuated and fenestrated endothelia. An occasional polymorphonuclear neutrophil was observed near these vessels in the corneal stroma in untreated (Fig. 1) and saline control nu/nu

FIG. 1. Central cornea of an untreated nu/nu mouse. A capillary (C) just beneath the epithelium (E) is composed of at least two endothelial cells and contains several erythrocytes (RBC) in its lumen. Within the corneal stroma, a keratocyte (K) exhibits a pale vesicular nucleus, a prominent nucleolus (Nu), as well as swollen mitochondria (M), and granular endoplasmic reticulum (ER). A portion of a neutrophil (PMN) is observed also. x5,400. FIG. 2. Central cornea of an intracorneally infected (108 bacteria) nu/nu mouse at 24 h. Two capillaries (C), one whose lumen contains a neutrophil (PMN) and the other whose lumen is devoid of blood cells, are found beneath the epithelium (E). A neutrophil (PMN), a phagocytic keratocyte (K), and intracellularly phagocytized bacteria (arrows) are observed also. x3,600. (a) Higher magnification of a cell fragment containing many phagocytized bacteria. The organisms appear to have undergone lytic changes. x11,250. 928

VOL. 22,1978

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FIG. 3. Fenestrated capillary located beneath the epithelium in the central cornea of an untreated nu/nu mouse. A pericyte (P) encircles the vessel whose lumen contains an erythrocyte (RBC) and several platelets (PI). Several large gaps (solid arrowhead) are observed within an endothelial cell and its surrounding pericyte. The normally thin basal lamina (BL) enclosing the vessel and pericyte shows a local thickening (arrow). Additionally, a bundle of collagen between the pericyte and the endothelium of the vessel also is observed (open arrowhead). x9,900. FIG. 4. Non-fenestrated capillary located beneath the epithelium in the central cornea of an untreated nu/nu mouse cornea. Two erythrocytes (RBC) are observed within the lumen of a small capillary (C). A thin basal lamina (BL) surrounds the endothelial cells forming the vessel wall. X9,450.

930

HAZLETT AND BERK

INFECT. IMMUN.

mally, one initially observes severe corneal opacity with heavy neutrophil infiltration in all experimentally infected mouse strains, even if they are naturally resistant strains which ultimately exhibit spontaneous recovery by bacterial clearance and restoration of corneal transparency (3). nu/nu mice, on the other hand, never exhibit corneal opacity with the iris not visible, nor is there as marked an increase in polymorphonuclear neutrophils at 24 h postbacterial infection, as observed in nu/+ as well as other mouse strains similarly infected (3). It is not currently known whether the athymic condition of nudes is directly responsible for the enhanced ocular response of these animals. However, one possible explanation for the heightened resistance and unusual corneal response to the bacteria in these nu/nu mice may be the fact that they possess subepithelial capillaries as well as an occasional polymorphonuclear neutrophil within the central cornea before experimental bacterial infection. These vessels may serve as the source both of soluble serum factors which enhance phagocytosis (4) and of migrating phagocytic cells. Despite these factors, numbers of neutrophils increase only slightly within nu/nu corneal capillaries or within the stroma 24 h after bacterial infection. Nevertheless, neutrophils, keratocytes, and their cell fragments contain intracellularly phagocytized and partially degraded bacteria, unlike similar cells of nu/+ mice which only rarely exhibit phagocytized bacteria. Perhaps then the presence of both blood vessels and neutrophils within the central corneas of nu/nu mice before bacterial infection act in concert to rapidly promote termination of the bacterial infection. Thus, nu/nu mice may be primed for rapid bacterial phagocytosis and intracellular bactericidal activity, unlike their nu/+ littermates, parent BALB/c (3), or other naturally resistant strains. Since the cellular response is wholly neutrophilic, it would appear that the resistance of nu/nu mice described herein is separate and distinct from an activated macrophage system. Although the evidence presented in this study suggests that blood vessels, neutrophils, and keratocytes play a role in the unusual heightened DISCUSSION resistance of nu/nu mice to experimental PseuThis study demonstrates that 1- to 2-month- domonas corneal infection, the exact mechaold congenitally athymic nu/nu mice, raised in nisms involved in resistance remain to be rea modified conventional environment, respond solved. At the present time it is not known in a markedly different manner and possess a whether this ocular resistance in the nude mouse heightened native resistance to experimentally is specific for only P. aeruginosa or whether it induced Pseudomonas ocular infection as com- extends to other organisms. pared with their nu/+ littermates, parent ACKNOWLEDGMENTS BALB/c (3), or other naturally resistant inbred This study was supported by Public Health Service grant mouse strains (manuscript in preparation). Nor-

mice. In infected nu/nu mice neutrophils appeared to have increased in number 24 h after bacterial infection only in the superior half of the corneal stroma and within the lumen of vessels located in this area (Fig. 2). The cytoplasm of neutrophils, keratocytes, or fragments of these cells contained intracellularly phagocytized and partially degraded bacteria, whereas the corneal stroma was devoid of organisms (Fig. 2 and 2a). In contrast, no bacteria or other foreign bodies were observed in the corneal stroma or within neutrophils or keratocytes in saline control or untreated nu/nu mice. In all nu/nu mouse corneas, no neutrophils were observed in the mid-to-deep stroma or near Descemet's membrane. Additionally, minimal stromal edema and an intact corneal endothelium were noted. In contrast, keratocytes throughout the stroma of all nu/nu mouse central corneas were edematous with pale, vesicular nuclei, prominent nucleoli, and swollen endoplasmic reticulum and mitochondria (Fig. 1). On the other hand, the central corneas of nu/+ littermates examined at 24 h after experimental bacterial infection showed numerous polymorphonuclear neutrophils (Fig. 5) and marked stromal edema, particularly in the midto-deep stroma (Fig. 6). Accumulations of neutrophils also were seen on both sides of Descemet's membrane (Fig. 7), whereas only an occasional neutrophil was found within the corneal epithelium. Descemet's membrane was denuded of endothelial cells and the basement membrane appeared ragged and thickened slightly (Fig. 7). Nu/+ mouse neutrophils, keratocytes or fragments of these cells, rarely contained phagocytized bacteria, and furthermore, both the corneal stroma and anterior chamber were devoid of organisms. Untreated and control corneas of nu/+ littermates lacked polymorphonuclear neutrophils and edematous keratocytes intrastromally (Fig. 8). Nor did experimental, untreated, or control central corneas of nu/+ littermates exhibit blood vessels before, or at 24 h after, experimental Pseudomonas infection or topical saline.

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FIG. 5. Central cornea of an intracorneally infected (10O bacteria) nu/+ mouse at 24 h. Neutrophils (PMN) are numerous in nu/+ mouse corneas when compared with nu/nu corneas similarly infected (see Fig. 2). Neutrophils accumulate beneath the epithelium (E) as well as in the mid-to-deep stroma. Intracellularly phagocytized and degraded bacteria are not often observed in nu/+ corneas. x3,780. In contrast, nu/nu corneas similarly treated show numerous intracellularly phagocytized and degraded bacteria (compare Fig. 5 with Fig. 2 and 2a). FIG. 6. Central cornea of an intracorneally infected (108 bacteria) nu/+ mouse at 24 h. Edema (arrows) is pronounced in the mid-to-deep stroma where neutrophils (PMN) are numerous. Collagen fibers (CF) are randomly oriented. x13,506). 931

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FIG. 7. Central cornea of an intracorneally infected (108 bacteria) nu/+ mouse at 24 h. Neutrophils (PMN) are observed within the anterior chamber (A). The cells (solid arrowhead) also are seen bordering Descemet's membrane (D) which is denuded of endothelial cells. x5,400. FIG. 8. Central cornea of a saline control nu/+ mouse at 24 h. The epithelium (E) and subjacent corneal stroma are well organized and show no edema. Two keratocytes (K) also are shown. x5,400.

VOL. 22, 1978

RESISTANCE OF nu/nu MICE TO P. AERUGINOSA

1 RO1 EYO 1935-01 from the National Eye Institute and in a grant from the Institute of Gerontology, Wayne State University. part by

1.

2. 3. 4.

LITERATURE CITED Cheers, C., and R. Waller. 1975. Activated macrophages in congenitally athymic "nude" mice. J. Immunol. 115: 844-847. Emmerling, P., H. Finger, and J. Bockemuhl. 1975. Listeria monocytogenes infection in nude mice. Infect. Immun. 12:437-439. Hazlett, L. D., D. Rosen, and R. S. Berk. 1976. Experimental eye infections caused by Pseudomonas aeruginosa. Opthal. Res. 8:311-318. Jutila, J. W. 1977. Congenitally athymic (nude) mice and

5. 6.

7. 8.

933

their application to the study of immunity and aging, p. 177. In T. Makinodan and E. Yunis (ed.), Comprehensive immunology. Immunology and aging. Plenum Medical Book Co., New York. Nickol, A. D., and P. F. Bonventre. 1977. Anomalous high native resistance of athymic mice to bacterial pathogens. Infect. Immun. 18:636-645. Pantelouris, E. M., and P. A. Fliach. 1972. Responses of athymic (nude) mice to sheep red blood cells. Eur. J. Immunol. 2:236-239. Rygaard, J. 1973. Thymus and self, immunobiology of the mouse mutant nude, p. 111-146. John Wiley and Sons, London (translated by J. N. Mitchell). Zinkernagel, R. M., and R. V. Blanden. 1975. Macrophage activation in mice lacking thymus-derived (T) cells. Experientia 31:591-593.

nu) mice to experimental Pseudomonas aeruginosa ocular infection.

Vol. 22, No. 3 INFECTION AND IMMUNITY, Dec. 1978, p. 926-933 0019-9567/78/0022-0926$02.00/0 Copyright i 1978 American Society for Microbiology Print...
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