Lipopolysaccharide Entry in the Damaged Cornea and Specific … · cornea via diffuse penetration...
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INFECTION AND IMMUNITY, 0019-9567/00/$04.0010 Mar. 2000, p. 1731–1734 Vol. 68, No. 3 Copyright © 2000, American Society for Microbiology. All Rights Reserved. Lipopolysaccharide Entry in the Damaged Cornea and Specific Uptake by Polymorphonuclear Neutrophils C. L. SCHULTZ, 1,2 A. G. BURET, 2,3 M. E. OLSON, 2,3,4 H. CERI, 2,3,4 R. R. READ, 3,4 AND D. W. MORCK 2,3 * Novavax, Rockville, Maryland, 1 and Department of Biological Sciences, 2 The Biofilm Research Group, 3 and Department of Microbiology and Infectious Diseases, 4 University of Calgary, Calgary, Alberta, Canada Received 12 October 1999/Accepted 30 November 1999 Bacterial lipopolysaccharide (LPS) is an important agent of induction of ocular pathology following corneal injury or wearing of contaminated contact lenses. The mechanism of LPS uptake through the corneal epithe- lium is unclear, and the role played by inflammatory cells in this phenomenon has not been previously assessed. Fluorescein isothiocyanate-labeled LPS from Escherichia coli was deposited onto the abraded corneas of New Zealand White rabbits. Epifluorescence microscopy of living excised corneas revealed diffuse LPS staining in the epithelial and stromal layers only in the vicinity of the abrasion. In addition, specific cellular uptake of LPS was suggested by fluorescence staining of cells along the abrasion site. In a second series of experiments, an anti-CD18 polyclonal antibody was used to block infiltration of polymorphonuclear neutro- phils (PMN) into the cornea. In these experiments, a diffuse distribution of fluorescent LPS was still observed along the abrasion, but the specific cellular uptake was abolished. The findings indicate that LPS enters the cornea via diffuse penetration at sites of injury and that specific cellular uptake of LPS occurs within the cornea via PMN which have migrated into the damaged tissue. Complications of common ocular diseases such as conjunc- tivitis, keratitis, ulceration, and general inflammation may re- sult in impaired visual function (1–3). Although bacterial col- onization of the eye clearly contributes to the pathogenesis of eye disease, these disorders may also result in the absence of culturable bacteria (1), and at least in part because of host defense factors. Such disorders may be associated with contact lens wear (1–3, 6, 7, 11, 13) or with specific surgical procedures (S. P. Holland, R. Mathias, D. W. Morck, and S. Slade, sub- mitted for publication). Indeed, overwhelming infiltration by polymorphonuclear neutrophils (PMN) is known to play a cen- tral role in the pathogenesis of tissue damage at numerous sites, including the eye (10). Yet, the role played by these host cells in the pathogenesis of ocular disease remains unclear. Also, while lipopolysaccharide (LPS) has been shown to induce corneal damage (10), the route of entry of free LPS into the corneal tissue has yet to be clarified. Bacterial LPS (endotoxin) can induce a variety of symptoms, including fever, reduction of blood pressure, inflammation, and tissue ulceration (8). LPS activates complement and ini- tiates the production of numerous cytokines (8, 9). Activation of these various responses, either individually or concurrently, may cause systemic and/or localized pathology (8). Indeed, induction of the complement system may lead to the produc- tion of anaphylatoxins (C3a and C5a) and cytokines such as interleukin-1, tumor necrosis factor alpha, and interleukin-2, which are potent proinflammatory mediators (8, 9, 14). These, in turn, amplify PMN recruitment at the sites of inflammation, hence contributing to the perpetuation of tissue injury (10). The objective of these experiments was to characterize the mechanisms of uptake of bacterial LPS at sites of corneal injury and to assess the role played by PMN in this phenom- enon. Adult New Zealand White rabbits (1.5 to 2.5 kg) were housed at the University of Calgary Life and Environmental Animal Resource Centre and provided commercial rabbit chow and water ad libitum. All animal procedures were carried out according to the guidelines of the Canadian Council of Animal Care and followed procedures approved by the Uni- versity of Calgary Animal Care Ethics Committee. Rabbits were anesthetized with halothane (4%; 2 liters per min). Cor- neas were abraded over an approximate length of 1 cm with a sterile 26-gauge needle from the medial canthus to the lateral canthus, with care being taken to limit the depth of abrasion of the epithelial layer, as described previously (4, 10). Following the abrasion, 10 ml of fluorescein isothiocyanate (FITC)-con- jugated LPS (1 mg/ml; from Escherichia coli O55:B5 (List Biological Labs Inc., Campbell, Calif.) was applied to the cor- neal surface with a 10-ml Hamilton syringe. At 15 min postin- oculation, the animals were euthanized with an overdose of sodium pentabarbital. Corneas were removed, rinsed in sterile phosphate-buffered saline (PBS), resuspended in sterile PBS, placed in 24-well sterile tissue culture plates (Nunc), and in- cubated at 37°C (5% CO 2 ) until being observed with an epi- fluorescence microscope, approximately 30 min postsurgery. IB4 antibody, a polyclonal antibody raised in rabbits against the PMN CD18 surface antigen, was a gift from John Wallace at the University of Calgary. This antibody is known to block CD18-dependent neutrophil extravasation (14). The antibody was delivered intravenously at a concentration of 1 mg of protein per kg of body weight in a 1-ml volume of sterile PBS, 18.25 h prior to surgery. One group of rabbits served as con- trols (n 5 6 eyes); a second group was treated with IB4 (n 5 6 eyes). In each animal, one eye was abraded while the con- tralateral side remained unmanipulated. Excised corneal prep- arations were observed with an inverted epifluorescence mi- * Corresponding author. Mailing address: University of Calgary, 2500 University Dr. NW, Calgary, Alberta, Canada T2N 1N4. Phone: (403) 220-5278. Fax: (403) 284-1537. E-mail: [email protected]. 1731 on August 15, 2020 by guest http://iai.asm.org/ Downloaded from
Transcript of Lipopolysaccharide Entry in the Damaged Cornea and Specific … · cornea via diffuse penetration...
INFECTION AND IMMUNITY,0019-9567/00/$04.0010
Mar. 2000, p. 1731–1734 Vol. 68, No. 3
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Lipopolysaccharide Entry in the Damaged Cornea and SpecificUptake by Polymorphonuclear Neutrophils
C. L. SCHULTZ,1,2 A. G. BURET,2,3 M. E. OLSON,2,3,4 H. CERI,2,3,4 R. R. READ,3,4
AND D. W. MORCK2,3*
Novavax, Rockville, Maryland,1 and Department of Biological Sciences,2 The BiofilmResearch Group,3 and Department of Microbiology and Infectious Diseases,4
University of Calgary, Calgary, Alberta, Canada
Received 12 October 1999/Accepted 30 November 1999
Bacterial lipopolysaccharide (LPS) is an important agent of induction of ocular pathology following cornealinjury or wearing of contaminated contact lenses. The mechanism of LPS uptake through the corneal epithe-lium is unclear, and the role played by inflammatory cells in this phenomenon has not been previouslyassessed. Fluorescein isothiocyanate-labeled LPS from Escherichia coli was deposited onto the abraded corneasof New Zealand White rabbits. Epifluorescence microscopy of living excised corneas revealed diffuse LPSstaining in the epithelial and stromal layers only in the vicinity of the abrasion. In addition, specific cellularuptake of LPS was suggested by fluorescence staining of cells along the abrasion site. In a second series ofexperiments, an anti-CD18 polyclonal antibody was used to block infiltration of polymorphonuclear neutro-phils (PMN) into the cornea. In these experiments, a diffuse distribution of fluorescent LPS was still observedalong the abrasion, but the specific cellular uptake was abolished. The findings indicate that LPS enters thecornea via diffuse penetration at sites of injury and that specific cellular uptake of LPS occurs within thecornea via PMN which have migrated into the damaged tissue.
Complications of common ocular diseases such as conjunc-tivitis, keratitis, ulceration, and general inflammation may re-sult in impaired visual function (1–3). Although bacterial col-onization of the eye clearly contributes to the pathogenesis ofeye disease, these disorders may also result in the absence ofculturable bacteria (1), and at least in part because of hostdefense factors. Such disorders may be associated with contactlens wear (1–3, 6, 7, 11, 13) or with specific surgical procedures(S. P. Holland, R. Mathias, D. W. Morck, and S. Slade, sub-mitted for publication). Indeed, overwhelming infiltration bypolymorphonuclear neutrophils (PMN) is known to play a cen-tral role in the pathogenesis of tissue damage at numeroussites, including the eye (10). Yet, the role played by these hostcells in the pathogenesis of ocular disease remains unclear.Also, while lipopolysaccharide (LPS) has been shown to inducecorneal damage (10), the route of entry of free LPS into thecorneal tissue has yet to be clarified.
Bacterial LPS (endotoxin) can induce a variety of symptoms,including fever, reduction of blood pressure, inflammation,and tissue ulceration (8). LPS activates complement and ini-tiates the production of numerous cytokines (8, 9). Activationof these various responses, either individually or concurrently,may cause systemic and/or localized pathology (8). Indeed,induction of the complement system may lead to the produc-tion of anaphylatoxins (C3a and C5a) and cytokines such asinterleukin-1, tumor necrosis factor alpha, and interleukin-2,which are potent proinflammatory mediators (8, 9, 14). These,in turn, amplify PMN recruitment at the sites of inflammation,hence contributing to the perpetuation of tissue injury (10).The objective of these experiments was to characterize the
mechanisms of uptake of bacterial LPS at sites of cornealinjury and to assess the role played by PMN in this phenom-enon.
Adult New Zealand White rabbits (1.5 to 2.5 kg) werehoused at the University of Calgary Life and EnvironmentalAnimal Resource Centre and provided commercial rabbitchow and water ad libitum. All animal procedures were carriedout according to the guidelines of the Canadian Council ofAnimal Care and followed procedures approved by the Uni-versity of Calgary Animal Care Ethics Committee. Rabbitswere anesthetized with halothane (4%; 2 liters per min). Cor-neas were abraded over an approximate length of 1 cm with asterile 26-gauge needle from the medial canthus to the lateralcanthus, with care being taken to limit the depth of abrasion ofthe epithelial layer, as described previously (4, 10). Followingthe abrasion, 10 ml of fluorescein isothiocyanate (FITC)-con-jugated LPS (1 mg/ml; from Escherichia coli O55:B5 (ListBiological Labs Inc., Campbell, Calif.) was applied to the cor-neal surface with a 10-ml Hamilton syringe. At 15 min postin-oculation, the animals were euthanized with an overdose ofsodium pentabarbital. Corneas were removed, rinsed in sterilephosphate-buffered saline (PBS), resuspended in sterile PBS,placed in 24-well sterile tissue culture plates (Nunc), and in-cubated at 37°C (5% CO2) until being observed with an epi-fluorescence microscope, approximately 30 min postsurgery.IB4 antibody, a polyclonal antibody raised in rabbits againstthe PMN CD18 surface antigen, was a gift from John Wallaceat the University of Calgary. This antibody is known to blockCD18-dependent neutrophil extravasation (14). The antibodywas delivered intravenously at a concentration of 1 mg ofprotein per kg of body weight in a 1-ml volume of sterile PBS,18.25 h prior to surgery. One group of rabbits served as con-trols (n 5 6 eyes); a second group was treated with IB4 (n 56 eyes). In each animal, one eye was abraded while the con-tralateral side remained unmanipulated. Excised corneal prep-arations were observed with an inverted epifluorescence mi-
* Corresponding author. Mailing address: University of Calgary,2500 University Dr. NW, Calgary, Alberta, Canada T2N 1N4. Phone:(403) 220-5278. Fax: (403) 284-1537. E-mail: [email protected].
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croscope (Zeiss Axiovert 25). The entire corneal surfaces wereassessed for LPS staining. Observations were recorded with aNikon 35-mm camera on 200-ASA Kodachrome film. Cellslabeled with FITC-LPS were counted by using the 103 mag-nification lens. Cell counts were expressed as the mean numberof cells per field of view under the 103 lens 6 the standarderror of the mean. Values were compared by one-way anal-ysis of variance and the Student Newman-Keuls test formultiple comparisons. P values ,0.05 were considered sig-nificant.
Results from microscopic observations are illustrated in Fig.1 and 2. Diffuse fluorescence was detected along the line ofabrasion for all preparations, but not in intact areas of thecorneal surface. In addition, within control animals not ex-posed to IB4, specific cells labeled with fluorescent LPS weredetected (Fig. 1). No staining was seen in nonabraded corneal
areas (data not shown). Intravenous IB4 treatment abolishedcell-specific FITC-LPS staining but did not alter the diffusestaining along the abrasion line (Fig. 2). As was observed incontrols, FITC-LPS staining was restricted to abrasion sites. Inall animals, no staining was detected on the nonabraded con-tralateral eye exposed to FITC-LPS and no specific uptakeof FITC-LPS by cells could be detected (data not shown).Cell counts were performed to determine the number ofcells specifically taking up FITC-LPS per field of view. Theresults of these FITC-LPS-labeled cell counts are illustrat-ed in Fig. 3. IB4 treatment abolished cell-specific FITC-LPSlabeling.
In summary, in an attempt to characterize the mechanism ofuptake of bacterial LPS into corneal tissue, this study investi-gated the effects of FITC-conjugated LPS deposition onto rab-bit corneas, in the presence or in the absence of epithelial
FIG. 1. A photomicrograph of an abraded rabbit cornea that was exposed to FITC-LPS immediately following abrasion. Note the diffuse staining of the cornealstroma adjacent to the abrasion and the concentration of fluorescence staining by cells within the corneal stroma or on the surface of the cornea. Magnification, 31,000.
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abrasion. The findings indicate that LPS enters the cornea onlyat sites of injury and that the uptake occurs via two distinctmechanisms: (i) via passive diffusion into the injured epithe-lium, and (ii) via specific cellular uptake. These results areconsistent with the intact corneal epithelium acting as a barrieragainst entry of proinflammatory soluble products. Using aCD18 antibody clearly assisted in the identification of the cellsresponsible for FITC-LPS uptake as PMN. These findings con-firm the observations of other investigators who demonstratedthat the PMN is the primary immune cell type infiltratingcorneal surfaces early in inflammation of the eye (4, 10). Datapresented here add to these observations evidence that thePMN acts as the sequestration cell for LPS that has enteredinto the cornea. PMN are responsible for the development ofcorneal marginal infiltrates and are a well-established feature
of the pathologic changes seen in diffuse lamellar keratitis(5, 12; S. P. Holland, R. Mathias, D. W. Morck, and S. Slade,submitted for publication) (Sands of the Sahara keratitis) fol-lowing laser-assisted in situ keratomileusis. Corneal marginalinfiltrates are known to occur as a result of direct bacterialinfection or following contact of the ocular surface with con-taminated medical devices, including contact lenses (7, 16, 17),and Sands of the Sahara keratitis (5, 12; S. P. Holland, R.Mathias, D. W. Morck, and S. Slade, submitted for publica-tion) is suspected to be caused by LPS contamination of lasersurgery sites. Findings from the present study demonstrate thatLPS alone may recruit PMN at sites of epithelial injury andhence may contribute to these syndromes. Since uncheckedPMN infiltration is directly responsible for tissue pathology ina number of inflammatory diseases (15), this study further
FIG. 2. A photomicrograph of an IB4 antibody-exposed, abraded rabbit cornea that was exposed to FITC-LPS immediately following abrasion. Note the diffusestaining along the corneal abrasion and the complete lack of specific cellular uptake of the FITC-LPS. Magnification, 31,000.
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underscores the significance of LPS contamination as a possi-ble contributor to specific ocular diseases. Experiments de-scribed herein lay the basis for future research into the basicmechanisms by which host defense factors may contribute tothe pathogenesis of such diseases.
This study was funded by The Natural Sciences and EngineeringResearch Council of Canada.
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Editor: J. D. Clements
FIG. 3. Cell counts from the abraded corneas of untreated FITC-LPS-ex-posed animals (Control) and the abraded corneas of IB4 antibody-treated,FITC-LPS-exposed animals. p, value is significantly lower than that of control(P , 0.05).
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