Effect of C3 Depletion on Experimental Pseudomonas aeruginosa ...

INFECTION AND IMMUNITY, Mar. 1984, p. 783-790

Vol. 43, No. 3

0019-9567/84/030783-08$02.00/0 Copyright © 1984, American Society for Microbiology

Effect of C3 Depletion on Experimental Pseudomonas aeruginosa Ocular Infection: Histopathological Analysis LINDA D. HAZLETT* AND RICHARD S. BERK

Departments of Anatomy and Immunology-Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201 Received 25 July 1983/Accepted 2 December 1983

The ocular histopathological response of C3-depleted and normal DBA/2J mice was compared after corneal scarification and experimental infection with topically applied Pseudomonas aeruginosa. Normal, non-C3-depleted mice were capable of mounting a vigorous polymorphonuclear leukocyte response at 24 h after bacterial challenge and eventually restored corneal clarity. In contrast, C3-depleted animals responded at 24 h to the ocular bacterial challenge with a decreased number of polymorphonuclear leukocytes migrating into the cornea. Additionally, the eyes of C3-depleted mice exhibited persistence of bacteria, cataract of the ocular lens, and loss of vision.

Corneal infection of susceptible strains of mice with Pseudomonas aeruginosa produces corneal opacity, endophthalmitis, and vision loss, frequently accompanied by panophthalmitis and phthisis bulbi. However, certain strains of mice are naturally resistant, and infected eyes do not undergo irreparable ocular damage. Rather, the animals clear the bacterial infection and restore corneal clarity (11). The important role of C3 complement in mediating resistance was determined recently by using cobra venom factor (CoVF) to deplete C3 and the terminal complement components in resistant DBA/2J mice (6; L. D. Hazlett and R. S. Berk, in Proceedings of an International Symposium on Immunology and Immunopathology of the Eye, in press). Either single or multiple injections of CoVF before scarification and topical bacterial infection of the eye rendered the mice susceptible. Complement involvement was found to occur early in the infectious process, and full lytic complement activity did not appear to be involved, since DBA/2J mice are genetically C5 deficient (14, 15). The purpose of the present report was to characterize histopathologically ocular P. aeruginosa infection in normally resistant and C3-depleted susceptible DBA/2J mice. The results show that C3-depleted animals, in contrast to normal mice, respond to bacterial infection with an initial delayed leukocytic mobility, bacterial persistence in the cornea, and subsequent loss of vision.

nearly undetectable. Levels remained low until day 4, when they again approached control values. Bacterial challenge. The organism used in these studies was P. aeruginosa ATCC 19660, which was found to be serum resistant by standard assay procedures (19). For bacterial challenge, mice were lightly anesthetized with ether and placed beneath a stereoscopic microscope, and the left eye was scarified (three 1-mm incisions) with a sterile 26gauge needle. Five microliters of a bacterial cell suspension containing approximately 1.25 x 108 viable organisms was delivered onto the scarified corneas with a micropipette with a sterile disposable tip (11). Corneas from both C3-depleted and nondepleted (normal) mice challenged in this manner were opaque 24 h later. Qualitatively, rating opacity on a scale of 0 (clear) to 4 (full opacity), nondepleted mice rated 3 or 4, whereas CoVF-treated mice rated 1 or 2 (slight opacity). Light and electron microscopy. At various times (days 1, 3, 5, 7, 10, 14, and 21 and 5 weeks) after infection, mice (three C3 depleted and three normal for each time period) were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg), and the challenged eye was enucleated. Each whole globe was fixed in a mixed fixative containing 1% osmium tetroxide and 2.5% glutaraldehyde in Sorenson phosphate buffer at pH 7.4 and 4° C for 3 h, dehydrated in graded ethanols, and embedded in Epon-Araldite. Sections (1.5-,um thick) were cut and stained with a modified Richardson stain (16). Representative sections were photographed with a Zeiss photomicroscope equipped with bright-field optics. For comparative purposes, all light micrographs were photographed and printed to a total magnification of x70. Light gold thin sections were cut, stained with aqueous uranyl acetate and lead citrate, observed, and photographed with a JEM-100CX or a Zeiss EM 9S-2 electron microscope, each operating at 60 kV.

MATERIALS AND METHODS Mice. Female DBA/2J mice were obtained from Jackson Laboratories (Bar Harbor, Maine). Mice were housed in plastic cages and fed laboratory rodent chow (RalstonPurina Co., St. Louis, Mo.) and water ad libitum. CoVF treatment. Partially purified CoVF was administered intraperitoneally at a dose of 200 U/kg (Cordis Laboratories, Miami, Fla.). Control mice received an equivalent volume of the appropriate diluent. Plasma C3 levels were monitored in Ouchterlony double diffusion gels as described previously (6). The kinetic pattern of C3 depletion was the same as that previously reported (6) (data not shown). Briefly, plasma C3 levels diminished at 2 h and by 6 h were *

RESULTS The response of C3-depleted and normal DBA/2J mice to corneal scarification and infection with P. aeriuginosa was characterized by light (Fig. 1 and 2) and transmission electron microscopy (Fig. 3 to 7) at various times after bacterial challenge. At 24 h after bacterial infection, normal mice (not C3 depleted) showed a heavy infiltration of polymorphonu-

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FIG. 1. Photomicrographs of the corneas of normal (A and C) and C3-depleted (B and D) DBA/2J mice at 24 h (A and B) and 3 days (C and D) after experimental P. aeruginosa-induced eye infection. Arrows: A, central epithelial defect; B, epithelial cell detachment; D, hyperemic iris. The arrowhead in D shows a region of bacterial aggregation. x70.

clear leukocytes (PMN) into the anterior ocular segment. PMN were particularly concentrated in the superficial half of the central corneal stroma, beneath a large epithelial defect. Descemet's endothelium was stripped from limbus to limbus, and corneal edema was significant (Fig. 1A). In contrast, the eyes of C3-depleted infected mice showed far fewer infiltrating cells in the anterior ocular segment. Cells were slightly more numerous in the peripheral corneal and conjunctival stroma than in the central cornea. Corneal

epithelium was intact from limbus to limbus, but in the central cornea, epithelial cell detachment from the underlying basal lamina had begun. Descemet's endothelium was partially stripped, and a few PMN adhered to Descemet's membrane. Corneal edema was slight (Fig. 1B). Three days after infection, PMN infiltration of the anterior segment remained heavy in normal DBA/2J mice, and corneal edema was marked. The iris was hyperemic, and both anterior and posterior synechia were seen (Fig. 1C). At 3 days after

VOL. 43, 1984

COMPLEMENT DEPLETION IN P. AERUGINOSA INFECTION

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FIG. 2. Photomicrographs of the corneas of normal (A and C) and C3-depleted (B and D) DBA/2J mice at 14 days (A and B) and 5 weeks (C and D) after experimental P. aeruginosa-induced eye infection. Arrows: A, fibroblasts; B, cataractous lens; C, blood vessel; D, anterior synechia. x70.

infection, the corneal epithelium of C3-depleted mice was stripped from limbus to limbus. PMN had increased in number, particularly in the mid-deep portions of the corneal stroma and in the anterior chamber. Areas of the superficial stroma of the central cornea were devoid of infiltrating phagocytic cells. In these regions, aggregates of bacteria clustered to form a thick band (Fig. 1D and 5). Descemet's endothelium was completely stripped limbus to limbus, and

the iris was hyperemic (Fig. 1D). Within 10 to 14 days after infection, normal DBA/2J mice began to show evidence of corneal healing, including reepithelialization, fewer infiltrating phagocytic cells, and fibroblast migration into the deep central corneal stroma (Fig. 2A). In contrast, the eyes of C3depleted mice exhibited numerous infiltrating phagocytic cells, anterior synechia, and central epithelial defect (Fig. 2B). Furthermore, at least 50% of the infected eyes exam-

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FIG. 3. Transmission electron micrograph of the central corneal stroma from a normal DBA/2J mouse 24 h after pseudomonas challenge. Heavy PMN infiltration of the corneal stroma is evident. A phagocytized organism (arrow) and a large phagocytic vacuole (V) are shown x 6,300.

ined stereomicroscopically and histologically showed cataract of the ocular lens. By 21 days to 5 weeks, non-C3depleted DBA/2J mice had essentially normal-appearing eyes. Few inflammatory cells were evident in the anterior segment, and only a few small blood vessels persisted in the normally avascular corneal stroma (Fig. 2C). On the other hand, the corneas of C3-depleted mice remained morphologically abnormal. The corneal epithelium appeared flattened and increased in density. Stromal blood vessels were numerous, and Descemet's membrane was tortuous and denuded. A few infiltrating phagocytic cells were present, and anterior synechia was common (Fig. 2D). Transmission electron microscopy substantiated and extended the light microscopic observations. PMN were con-

firmed as the predominant infiltrating phagocyte in the stroma of non-C3-depleted mice at 24 h after bacterial infection. Organisms were seen within PMN cytoplasm, and many cells had large phagolysosomal vacuoles. Stromal keratocytes were degenerated, with only remnants of cells remaining. No free bacteria were observed in the collagenous corneal stroma (Fig. 3). In contrast, the stroma of C3depleted mice exhibited only a paucity of infiltrating phagocytic cells at 24 h after infection. PMN were the predominant cell type, and only an occasional macrophage was observed. Few PMN contained either large phagocytic vacuoles or organisms at this time, and free organisms were seen within the collagenous corneal stroma (Fig. 4). By 3 days after infection, the corneal stroma of normal DBA/2J mice was

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COMPLEMENT DEPLETION IN P. AERUGINOSA INFECTION

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filled with necrotic PMN. No free organisms were seen. In contrast, C3-depleted mice at both 3 and 5 days after infection exhibited numerous free bacteria in the superficial (Fig. 5) and mid-deep (Fig. 6) corneal stroma. At these times, PMN were present in the anterior chamber and within the mid-deep corneal stroma but were absent from the superficial central cornea. In the mid-deep corneal stroma, PMN from C3-depleted mice often contained numerous, rather intact-appearing organisms (Fig. 7). As early as 3 weeks after the initial infection, the corneal epithelium and endothelium of mice not C3 depleted appeared morphologically near normal. In contrast, corneas from C3-depleted mice did not appear normal even at 5 weeks after infection. The corneal epithelial surface of these eyes showed adherent bacteria at 2

to 5 weeks after the experimental infection. In addition, Descemet's endothelium failed to resurface the denuded

membrane, and stromal disorganization of collagen persisted. DISCUSSION In previous studies, it has been shown that natural resistance to corneal infection with P. aeruginosa is genetically controlled (1, 3, 4; Berk et al., in Proceedings of an International Symposium on Immunology and Immunopathology of the Eye, in press). Furthermore, it was found that at least one factor which contributes to this natural resistance is the activity of complement, especially the C3 compo-

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