Cytokine Modulation of Human Corneal Epithelial Cell ICAM-1 (CD54) Expression

Exp. Eye Res. (1998) 67, 383–393 Article Number : ey980514

Cytokine Modulation of Human Corneal Epithelial Cell ICAM-1 (CD54) Expression J U D I T H Y A N N A R I E L LO-B R O WNa,c, C S I L L A K. H A L L B E R Ga, H E L E N E H A= B E R LE d † , M I R I A M M. B R Y S Kb,c, Z I L I J I A N Gd, J A N A K A. P A T E Ld, P E T E R B. E R N S Td,e    S T E F A N D. T R O C M Ea* Departments of a Ophthalmology and Visual Sciences, b Dermatology, c Human Biological Chemistry and Genetcs, d Pediatrics, and the e Sealy Center for Molecular Science, University of Texas Medical Branch, Galverston, TX 77555–0787, U.S.A. (Received Columbia 25 November 1997 and accepted in revised form 23 March 1998) To determine whether pro-inflammatory cytokines modulate intercellular adhesion molecule-1 (ICAM1 ; CD54) expression on cultured primary human corneal epithelial cells (HCEs), confluent HCEs were treated with various concentrations of interferon-γ (IFN-γ), interleukin-1α (IL-1α), IL-1β, IL-4, tumor necrosis factor-α (TNF-α), or combinations over time. ICAM-1 expression was measured by flow cytometry and\or a cell-based ELISA using a monoclonal mouse anti-human CD54 antibody. The apparent MW of ICAM-1 protein was determined by immunoprecipitation of biotinylated HCEs. RT-PCR was used to detect ICAM-1 RNA. The mature cell surface form of HCE ICAM-1 was " 110 kDa as determined by immunoprecipitation. IFN-γ and TNF-α induced both dose- and time-dependent increases in ICAM-1 expression. An " 20-fold increase in ICAM-1 was seen at 50–100 U IFN-γ ml−". ICAM-1 specific mRNA accumulated " 4n5-fold after IFN-γ treatment. TNF-α (100 U ml−") induced a consistent " 6n0-fold increase in ICAM-1 expression. When IFN-γ and TNF-α were mixed, at sub-optimal concentrations of each, a synergistic effect on ICAM-1 expression was not detected. Neither IL-4, IL-1α nor IL-1β affected ICAM-1 expression in a consistent fashion. In summary, ICAM-1 was modulated on primary human corneal epithelial cells by the cytokines IFN-γ and TNF-α in a dose- and time-dependent fashion. Cytokine modulation of corneal epithelial cell ICAM-1 during inflammation may contribute to corneal epithelial cell injury by aiding the attachment of inflammatory cells such as eosinophils which express the receptor for ICAM-1, the β integrins (CD11a,b,c\CD18). # 1998 Academic Press # Key words : CD54 ; ICAM-1 ; human corneal epithelium ; cytokines ; allergy ; inflammation.

1. Introduction The migration of effector cells to a site of inflammation depends on the presence of adhesion receptors and counter receptors on the immune cells, endothelium, extracellular matrix, and epithelium (Leff, Hamann and Wegner, 1991). Leukocyte-epithelial cell interactions are mediated, in part, by intercellular adhesion molecule-1 (ICAM-1 ; CD54) and its receptors the β # integrins (CD11a,b,c\CD18). ICAM-1 is a member of the immunoglobulin supergene family, is expressed constitutively on all leukocytes, is also up-regulated on the endothelium and epithelium by pro-inflammatory cytokines (Dustin et al., 1986 ; Leff, Hamann and Wegner, 1991) ; the β integrins are present on all # leukocytes including eosinophils (Dustin et al., 1986, 1988 ; Rothlein et al., 1986). ICAM-1 is an important adhesion molecule for tracheal epithelial-eosinophil attachment and degranulation (Leff, Hamann and Wegner, 1991), T lymphoblast-epidermal keratinocyte * Address all reprint requests to : Stefan D. Trocme, M.D., Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, TX 77555–0787, U.S.A. † Present address : Universita$ tsklinik fur Anaesthesiologie und Transfusionmedizin, Eberhard-Karls-Universita$ t Tu$ bingen, HoppeSeyler-Str. 3, 72076 Tu$ bingen.

0014–4835\98\100383j11 $30.00\0

interactions in vitro (Dustin et al., 1986, 1988), and inflammatory cell infiltration into the eye during an acute ocular inflammatory response (Whitcup et al., 1995). Among its many roles, ICAM-1 is thought to play a pivotal role in inflammation associated with allergic reactions and is considered to be a marker of allergic inflammation (Canonica et al., 1994, 1995). Bronchial biopsies from patients with allergic asthma expressed statistically higher levels of ICAM-1 on the epithelium compared to normal controls or to nonallergic asthmatics. Interestingly, the non-allergic asthmatics did not have significantly elevated ICAM-1 compared to normal controls (Gosset et al., 1995). ICAM-1 expression on the conjunctival epithelium can also be modulated after topical allergen provocation in allergic human subjects (Canonica et al., 1995 ; Ciprandi et al., 1993) and by proinflammatory cytokines in vitro (Paolieri et al., 1997), supporting a role for ICAM-1 during inflammation associated with ocular allergy. Using immunohistochemistry, our laboratory has recently demonstrated the presence of ICAM-1 expression on the epithelium from an ulcerated cornea, during a severe ocular allergic response. A dramatic up-regulation in ICAM-1 expression was detected on the basal and middle corneal epithelium # 1998 Academic Press

384

on a specimen from a vernal keratoconjunctivitis (VKC) patient when compared to normal control tissue (Gill et al., 1996). Since eosinophils have been implicated as a major effector cell in severe ocular allergic responses leading to corneal ulceration as seen in VKC (Trocme and Aldave, 1994 ; Trocme et al., 1989), the up-regulation of ICAM-1 on affected corneal tissue in VKC suggests an important mechanistic role for ICAM-1 in this disease. However, little is known of the mechanisms controlling ICAM-1 regulation in the corneal epithelium. Several cytokines are present at sites of allergic inflammation in a variety of tissues and have been implicated in ICAM-1 regulation. The complement of cytokines present and functional in the eye during ocular allergy is not as well studied. While some information can be found in the literature, other pieces of information can be inferred from the cell types known to be present in the eye and examining the cytokines they elaborate when in other tissues. For example, T-lymphocytes of the Th-2-type are thought to be the predominant type present in the conjunctiva in VKC (Maggi et al., 1991). These cells produce IL-3, -4, and -5 and GM-CSF, TNF-α, among others (Del Prete, 1992). Mast cells, present in the conjunctiva in all forms of ocular allergy (Foster, 1995), are known to produce IL-1, -3, -4, and 5, GM-CSF, TNF-α, and IFN-γ (Galli, Gordon and Wershil, 1993 ; Galli and Wershil, 1996 ; Harvima et al., 1994). Eosinophils are present in the conjunctiva and in some cases, cornea of ocular allergy patients (Trocme and Aldave, 1994 ; Trocme et al., 1993) and are known to produce IL-8, RANTES, MIP-1 α, IL-3, -4, -5, -6, IL-1, and TNF-α (Nakajima, Gleich and Kita, 1996). The purpose of the present study was to investigate the regulation of corneal epithelial ICAM-1 by the pro-inflammatory and immunoregulatory cytokines IFN-γ, TNF-α, IL-1 α, IL-1β and IL-4. 2. Materials and Methods A murine monoclonal antibody to human ICAM-1 (subclass IgG ) was purchased from Pharmingen (San " Diego, CA, U.S.A.) and used for immunoprecipitations, flow cytometry, and the ELISA. Protein A conjugated to Sepharose beads, casein blocking solution, and NHS-biotin were purchased from Pierce (Rockford, IL, U.S.A.). Human recombinant TNF-α and IFN-γ were purchased from Genzyme (Cambridge, MA, U.S.A.). Human recombinant IL-1β and IL-1α were purchased from R & D Systems (Minneapolis, MN, U.S.A.). Human recombinant IL-4 was from Pharmingen (San Diego, CA, U.S.A.). Isotype control murine monoclonal antibody (IgG ) was purchased from Dako Corp. " (Carpinteria, CA, U.S.A.). Goat anti-mouse IgG conjugated to alkaline phosphatase or horse radish peroxidase was purchased from Sigma (St. Louis, MO, U.S.A.) and Chemicon (Temecula, CA, U.S.A.), respectively. Gel electrophoresis reagents were pur-

J. Y A N N A R I E L LO-B R O W N E T A L.

chased from Sigma or Novex (San Diego, CA, U.S.A.). Bovine serum albumin (BSA), and O-phenylenediamine were purchased from Sigma. Keratinocyte Growth Medium supplemented with bovine pituitary extract (KGM\BPE) was from Clonetics (San Diego, CA, U.S.A.). Trypsin\EDTA was from Gibco (Gaithersburg, MD, U.S.A.). The fibronectin-collagen coating mixture was from BRFF (Ijamsville, MD, U.S.A.). Nitrocellulose sheets (0n1 µm) were from Schleicher and Schuell (Keene, NH, U.S.A.). The Trizol reagent was from Life Techologies (Gaithersburg, MD, U.S.A.). The ICAM-1 and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) PCR primers were from Stratagene (La Jolla, CA, U.S.A.) and Clontech (Palo Alto, CA, U.S.A.), respectively. GeneAmp RNA PCR kit was purchased from Perkin Elmer Cetus (Norwalk, CT, U.S.A.). Human Corneal Epithelial Cell Isolation and Culture Epithelial cultures were established from human corneal buttons unsuitable for transplantation (Lions Eye Banks of Houston, San Antonio and Austin, TX, U.S.A.). the use of human tissue was approved by the Institutional Review Board at the University of Texas Medical Branch at Galveston. Corneal buttons were cut from Optisol preserved human corneal tissue using Castroviejo curved corneal scissors, incubated in 0n25 % pronase and 0n25 % trypsin in Ca#+ and Mg#+ free Dulbecco’s phosphate buffered saline (DPBS) for 1 hr at 37mC, fetal bovine serum was then added to neutralize the enzymes. Detached epithelial cells were washed twice with DPBS, then cultured with KGM\BPE in tissue culture flasks previously coated with a fibronectin-collagen mix. Cells from a minimum of 8 corneas were pooled before plating. The epithelial origin of these cultures was confirmed by immunohistochemistry using the monoclonal anti-cytokeratin antibodies AE1\AE3. Once the cells had grown to confluency, they were passaged at a 1 : 2 ratio after being detached with 0n05 % trypsin\EDTA ; all experiments utilized cells at passage 2 only. HCEs were grown to confluency in either culture flasks or 96-well plates, then treated with various concentrations of IFN-γ (1–500 U ml−"), IL-1α or -β (0n001–10 ng ml−"), IL-4 (1–20 ng ml−"), TNF-α (25–400 U ml−"), or various combinations of each over time. Flow Cytometry After cytokine treatment, corneal epithelial cells were non-enzymatically released from the substrate by incubating in 10 m EDTA in phosphate buffered saline (PBS) for 30 min at 37mC, then gently scraping with a disposable cell scraper (Costar). One millilitre of cells (5i10&) was then incubated in suspension with a mouse anti-human ICAM-1 monoclonal antibody at a 1 : 200 dilution at 4mC for 30 min. The cells were washed twice in PBS, then incubated with goat anti-

C O R N E A L E P I T H E L I A L I C AM-1 E X P R E S S I ON

385

(A)

(B)

F. 1(A). Modulation of ICAM-1 expression on HCEs by IFN-γ as determined by flow cytometry. HCEs were incubated in the absence or presence of various concentrations of IFN-γ for 48 hr. The cells were removed with EDTA and gentle scraping, fixed, then stained for ICAM-1 with a mouse anti-human ICAM-1 antibody or an isotype matched IgG control antibody followed by a fluorescein-conjugated anti-mouse secondary antibody. Fluorescence intensity was measured using a FACscan. Shown are the data for the isotype control antibody on cells stimulated with 50 U IFN-γ ml−", ICAM-1 staining in the unstimulated control cells, and ICAM-1 staining in cells stimulated with 50 U IFN-γ ml−" (B) shows the number of fluorescent cells for each IFN-γ concentration tested (percent of the total sample).

mouse IgG conjugated to fluorescein isothiocyanate at 4mC for 30 min. The cells were washed twice in PBS, fixed in 2 % paraformaldehyde, incubated for 30 min with 10 µg ml−" DNase in PBS at 4mC to minimize cell clumping, then analysed for fluorescence intensity by single-color flow cytometry (FACScan ; Becton Dickinson). A nonreactive, isotype-matched mouse monoclonal IgG antibody was included as a negative control to measure background fluorescence. Epithelial cells were gated based on forward and side scatter and, subsequently, the relative expression of ICAM-1 was measured on the population. The mean and percent fluorescence of 1i10% cells were determined for each antibody.

ELISA for Detecting ICAM-1 Expression on HCEs The ICAM-1 ELISA is based on the protocols of Rothlein et al. (Rothlein et al., 1988) and Patel et al. (Patel et al. 1995) ; minor modifications were implemented to adapt the technique to HCEs. HCEs were grown to confluency on fibronectin\collagen-coated 96-well round-bottomed plates (Corning, Corning, NY, U.S.A.). The monolayers were fixed in 1 % paraformaldehyde in PBS, endogenous peroxidase activity quenched with NH Cl, then non-specific % binding sites were blocked in PBS containing casein, Tween 20, and BSA. The wells were incubated with a mouse IgG MAb to human ICAM-1 or an isotype control, followed by a goat anti-mouse IgG secondary antibody conjugated to horse radish peroxidase. The wells were washed with PBSj0n5 % Tween 20, then

incubated with O-phenylenediamine and H O in # # citrate buffer. Color development was measured on an ICN Multiskan MCC\340 ELISA reader (Costa Mesa, CA, U.S.A.) at 492 nm. Non-treated control cells were also assayed for ICAM-1 expression at each time point. Data points were determined in triplicate. The relative amount of ICAM-1 was calculated by first subtracting non-specific binding of the isotype-matched control antibody from all test values, then experimental data was calculated as the percent of the non-treated control for each time point [meanpstandard deviation (..)]. Immunoprecipitation of ICAM-1 from SurfaceBiotinylated HCEs Confluent HCEs were treated with IFN-γ for 48 hr to induce ICAM-1 expression, the cell layers were then washed 3 times in PBS at room temperature. NHSbiotin was dissolved in DMSO at a final concentration % of 20 mg ml−", 2n5 µg of which was then added to 15 ml PBSj10 m EDTA. Five millilitres of the mixture was added\25 cm# of growth area at 23mC for 2 hr with gentle rocking. The cells were washed 3i in cold PBS, then solubilized in lysis buffer (PBS pH 7n5, 0n5 % Triton X-100 (v\v), 10 m EDTA, 2 m diisopropylfluorophosphate, 1n5 m phenylmethylsulfonylfluoride, and 1n5 m N-ethylmaleimide). The anti-ICAM-1 and isotype control mouse MAbs were individually complexed with Protein A-Sepharose beads, washed in PBS, then in Buffer 1jBSA [10 m HEPES pH 7n5, 140 m NaCl, 1n5 % BSA (w\v)] to block non-specific binding sites, then again in PBS.

386

J. Y A N N A R I E L LO-B R O W N E T A L.

F. 2. Modulation of ICAM-1 expression on HCEs by IFN-γ as determined by ELISA. HCEs were grown to confluency in 96 well plates then incubated in the absence or presence of various concentrations of IFN-γ for 24, 48, and 72 hr. After fixation and blocking of non-specific binding sites, ICAM-1 was quantitated by incubating with a mouse anti-human ICAM-1 MAb, followed by a goat anti-mouse IgG conjugated to horse radish peroxidase. The data are presented as the percent of the nontreated control for each particular time point (meanp..). The insert shows ICAM-1 expression on HCEs incubated in the absence or presence of 100 U IFN-γ ml−" for 3, 6, 24, 48, and 72 hr.

The Triton X-100 in the cell lysate was diluted to 0n1 % with ice cold PBS, then the cell lysate was incubated consecutively with first, the isotype control-Protein ASepharose-conjugate, then the ICAM-1-Protein ASepharose-conjugate for 2 hr at 4mC with gentle rocking. Both sets of beads were washed individually 3 times in PBS, the bound material was eluted in 1i sample buffer, then separated using non-reducing SDS-PAGE (8n5 %) according to Laemmli (Laemmli, 1970). The proteins were then transferred to a nitrocellulose membrane as described (YannarielloBrown, 1990), and non-specific binding sites on the nitrocellulose were blocked in 25 m Tris pH 7n2, 200 m NaCl, 0n01 % Tween-20 (v\v), and 5 % milk solids (w\v). Biotinylated ICAM-1 was visualized by incubating the nitrocellulose with streptavidin-alkaline phosphatase, then developed with the appropriate chromogen in citrate buffer. Detection of ICAM-1 Specific mRNA using RT-PCR HCEs were grown to confluency then treated with either KGM\BPE alone (unstimulated control) or 100 U IFN-γ ml−" KGM\BPE for 48 hr. The cell layers were washed 3 times in PBS then extracted with the

Trizol reagent and total RNA isolated according to manufacturers instructions. A GeneAmp RNA PCR Kit was used to perform the reverse transcription and polymerase chain reaction on total RNA as described (Patel et al., 1995). cDNA was generated using Moloney murine leukemia virus reverse transcriptase and oligo d(T) primers, then " 50 µg cDNA was incubated with 0n15 m of either the ICAM-1 or G3PDH primers in separate PCR reaction mixtures initially with hot start at 95mC for 2 min, then for 35 cycles at 95mC for 45 sec, 60mC for 30 sec, and 72mC for 1 min, followed by a final amplification at 72mC for 10 min in a DNA thermal cycler (Perkin Elmer Cetus). The resulting PCR products were separated on a 6 % polyacrylamide gel using ØX174 DNA as a standard, visualized with ethidium bromide staining (0n5 mg ml−"), then photographed with Polaroid 55 film. Quantitation of ban intensity was performed on the negatives using a LYNX 5000 Digital Image Analysis System (Applied Imaging, Santa Clara, CA, U.S.A.). The start for the ICAM-1 sense primer is at base 238 and the stop for the antisense primer is at 636 ; the expected size of the ICAM-1 specific PCR product is 398 bases. The size of the expected G3PDH control PCR product is 983 bases.

C O R N E A L E P I T H E L I A L I C AM-1 E X P R E S S I ON

387

Statistical Analysis All experiments were performed in triplicate. The data is expressed as the meanp.. of the sample population. Standard deviation bars were included for all data points. However, in some cases they were smaller than the symbols used and cannot be seen. Analysis of variance was performed using Systat 6.0 for Windows (Chicago, IL, U.S.A.) and Student’s t test using Statview 4.0 for Macintosh (Abacus Concepts, CA, U.S.A.). Data were considered significant if P 0n05. 3. Results IFN-γ Modulates ICAM-1 Protein and mRNA Expression in Cultured Primary HCEs The effect of IFN-γ on ICAM-1 expression on primary HCEs was investigated using both standard flow cytometry and a cell-based ELISA. Both techniques gave identical results. HCEs were incubated with increasing concentrations of IFN-γ for 48 hr before determining ICAM-1 expression via flow cytometry. Figure 1(A) shows the relative degree of ICAM1 expression using 50 U IFN-γ ml−" only. Figure 1(B) shows the number of cells positive (% of the nontreated control) for ICAM-1 after treating with 1, 10 or 50 U IFN-γ ml−". A low level of constitutive ICAM-1 expression was consistently detected in unstimulated control cells, representing " 5 % of the total cell population. A linear increase in the percent positive cells was seen with increasing concentrations of IFNγ. After stimulation with 50 U IFN-γ ml−" for 48 hr,
90 % of the total cell population demonstrated some degree of ICAM-1 expression. Additional details of ICAM-1 regulation were evaluated by ELISA. HCEs were cultured to confluency in 96-well plates, then treated with various concentrations of IFN-γ over time. IFN-γ induced a significant dose and time-dependent up-regulation of ICAM-1 expression compared to unstimulated controls (Fig. 2). Maximal stimulation was seen at 50 U ml−" at 72 hr, in which ICAM-1 expression was " 20-fold over control values. In all experiments, ICAM-1 expression was barely detectable at 3 hr post-stimulation using 100 U IFN-γ ml−" (126p38 % of control ; n l 4 experiments ; see insert), but could be readily detected by 6 hr (249p47 % of control ; n l 4 experiments). To determine the molecular weight of cell surface HCE ICAM-1 and confirm the specificity of the mouse monoclonal anti-human ICAM-1 MAb, immunoprecipitations of surfaced-biotinylated HCEs were performed. Before immunoprecipitating ICAM-1, HCEs were first stimulated for 48 hr with 100 U IFN-γ ml−" to increase surface ICAM-1 expression. The multiple band pattern seen in the lane containing starting material reflects the successful biotinylation of HCE proteins (Fig. 3, lane C). Immunoprecipitated ICAM-1 displayed an apparent Mr " 110 000 (lane A). These

F. 3. The molecular weight of HCE cell surface ICAM-1 is " 110 000 as determined by immunoprecipitation. After stimulating with 100 U IFN-γ ml−", the cell surface of live intact HCEs was biotinylated using NHS-biotin. The cell layers were solubilized, then immunoprecipitated with a mouse anti-human ICAM-1 MAb and an isotype-matched mouse control MAb. The immunoprecipitates were separated using non-reducing SDS-PAGE (8n5 %), transferred to nitrocellulose, then biotinylated ICAM-1 was visualized by reacting with streptavidin-AP and the appropriate color developing reagents. MW of the standards are shown in kDa. Lane A contains the immunoprecipitate obtained from cell extracts with the anti-ICAM-1 MAb ; Lane B contains the immunoprecipitate obtained using the isotype control MAb ; and Lane C contains an aliquot of the biotinylated starting material (1 µg total protein).

data agree with previously published molecular weight determinations for ICAM-1 from other cell types. These values ranged from 76–114 kDa, depending on the cell type used ; presumably, this is due to variability in the degree of post-translational modifications

388

F. 4. Detecting ICAM-1 specific mRNA using RT-PCR. The expression of ICAM-1 RNA was determined in nonstimulated control cells (k) and cells treated with 100 U IFN-γ ml−" for 48 hr (j). A band corresponding to " 400 bases was detected in IFN-γ treated cells using ICAM-1 specific primers (see Methods). As a control to ensure equal sample loading, primers for the control gene G3PDH were also used. Band intensities were measured by image analysis and the ratio of the ICAM-1 band intensity to the G3PDH band intensity is shown.

(Carlos and Harlan, 1994). The isotype control antibody did not immunoprecipitate any significant proteins (lane B).

J. Y A N N A R I E L LO-B R O W N E T A L.

To examine whether ICAM-1 RNA was modulated in HCEs after IFN-γ treatment, cells were incubated with either medium alone or 100 U IFN-γ ml−" for 48 hr. Total HCE RNA was isolated, then ICAM-1 mRNA was detected using ICAM-1 specific primers and RT-PCR. The results from one representative experiment is shown in Fig. 4. A band corresponding to " 400 bp was detected in IFN-γ treated cells, as expected for the particular primers used. ICAM-1 specific bands were barely discernible in the control cells under these conditions. The fact that equal amounts of cDNA were used in each sample was verified by control G3PDH primers and image analysis of the resulting bands. The ratio of ICAM-1 PCR product to the G3PDH control was 0n07 and 0n33 in the control cells and IFN-γ treated cells, respectively. This represents a significant " 4n5-fold increase over non-stimulated cells. TNF-α Modulates ICAM-1 Expression on Cultured Primary HCEs When HCEs were incubated with increasing concentrations of TNF-α, a significant dose- and timedependent increase in ICAM-1 expression was seen (Fig. 5). An increase in ICAM-1 protein could be

F. 5. Modulation of ICAM-1 expression on cultured HCEs by TNF-α. HCEs were incubated with various concentrations of TNF-α over time. Non-treated control cells were also assayed for ICAM-1 expression at each time point. The data were calculated as in Fig. 2. ICAM-1 expression increased in a dose and time-dependent manner. The maximal response was seen at 24 hr with no further increase with time and plateaued at 200 U ml−".

C O R N E A L E P I T H E L I A L I C AM-1 E X P R E S S I ON

389

F. 6. A mixture of TNF-α and IFN-γ modulated HCE ICAM-1 expression in a non-synergistic fashion. HCEs were incubated with a mixture of sub-optimal concentrations of TNF-α (100 U ml−") and IFN-γ (1 U ml−") over time. Non-treated control cells were also assayed for ICAM-1 expression at each time point. The data were calculated as in Fig. 2, then the control value was subtracted to show only increases over control values. Hence, the data is expressed in relative units.

T I Effects of IL-1α, IL-1β, and IL-4 on HCE ICAM-1 expression Interleukin IL-1α (10 ng ml−") IL-1β (10 ng ml−") IL-1αjβ (5 ng each ml−") IL-4 (20 ng ml−")

3 Hours

6 Hours

24 Hours

48 Hours

72 Hours

75p26

133p53

144p38

134p19

184p22

91p16

123p22

183p44

122p17

102p7

84p12

92p16

137p18

125p20

100p7

128p39

125p46

117p63

144p60

186p35

HCEs were incubated with 10 ng ml−" of IL-1α, or IL-β, or a 1 : 1 mixture of IL-1αjIL-1β (5 ng ml−" each), 20 ng IL-4 ml−", over time. Nontreated control cells were also assayed for ICAM-1 expression at each time point. The data were calculated as in Fig. 2 and represent the mean of 3 separate experiments performed in triplicate in the case of IL-1α and IL-1β, and 2 separate experiments for IL-4.

detected by 6 hr post-stimulation. Maximal stimulation was seen at 200 U TNF-α ml−", where ICAM-1 levels were " 6-fold above control values. ICAM-1 levels plateaued at 24 hr and remained constant up to 48 hr. However, detectable ICAM-1 protein actually declined to control values 72 hr post-stimulation (not shown). This phenomenon was consistently seen in 3 separate experiments.

To determine if adding TNF-α and IFN-γ simultaneously could affect HCE ICAM-1 expression in a synergistic fashion as described for other epithelia (Barker et al., 1990 ; Bloemen et al., 1993 ; Dustin et al., 1988). TNF-α and IFN-γ were added simultaneously in a 100 : 1 ratio in which TNF-α and IFNγ were kept constant at 100 U ml−" and 1 U ml−", respectively. ICAM-1 expression was then measured

390

over time. ICAM-1 expression increased over time in a non-synergistic, fashion (Fig. 6). ICAM-1 protein was also measured after stimulating cells with a 1 : 1 ratio of TNF-α and IFN-γ (100 U each ml−"), no additional ICAM-1 expression was detected above that seen for IFN-γ alone (not shown).

Lack of ICAM-1 Modulation on Cultured Primary HCEs by IL-1α, IL-1β, or IL-4 Cultured HCEs were treated with various concentrations of IL-1α, IL-1β, or a 1 : 1 mixture of both (5 ng ml−" each), then examined for ICAM-1 expression using the ELISA. Neither IL-1α alone, IL-1β alone, nor a 1 : 1 mixture of both induced a consistent upregulation of ICAM-1 expression on HCEs at the concentrations tested even after 72 hr of stimulation. The results of only the highest concentration of cytokine used are shown in Table I. A statistically significant effect was seen only at 10 ng IL-1α ml−" at 72 hr and at 10 ng IL-1β ml−" at 24 hr. In both cases the effect was nominal, being 2-fold in both cases (184p22 and 184p44 % of control for IL-1α and IL1β, respectively). To ensure that the lots of IL-1α and -β used were indeed biologically active, A549 cells were incubated with the same lot of IL-1α or -β ; A549 cells are known to up-regulate ICAM-1 in response to IL-1α and -β (Patel et al., 1995). An increase in ICAM1 expression was detected on these cells using the ELISA (not shown), indicating that the cytokines are indeed biologically active and the lack of response is a function of the HCEs and not the reagents used. The lack of response to IL-1 is not likely to be due to a lack of IL-1 receptors on HCEs since incubating HCEs with IL-1α results in the release of RANTES from HCEs (Trocme et al., 1997). A statistically significant effect of IL-4 on ICAM-1 expression was detected only at 20 ng ml−" at 72 hr post-stimulation (186p35 % of control). No additional effects were detected at any time point when IL-4 was used in combination with TNF-α (not shown).

4. Discussion Studies were conducted to determine if pro-inflammatory and immunoregulatory cytokines modulate the expression of the cell adhesion protein ICAM-1 (CD54) on human corneal epithelial cells in vitro. Using both flow cytometry and a cell-based ELISA, a low but consistent level of constitutive ICAM-1 expression was detected in unstimulated control HCEs. This observation was also made by others using flow cytometry (Iwata et al., 1997). While ICAM-1 has been shown to be constitutively expressed on organcultured mice corneal epithelium at relatively low levels (Hobden et al., 1995), constitutive expression of ICAM-1 has not been shown on human corneal

J. Y A N N A R I E L LO-B R O W N E T A L.

epithelium in vivo (Gill et al., 1996 ; Goldberg, Ferguson and Pepose, 1994 ; Iwata et al., 1997 ; Philipp, 1994 ; Whitcup et al., 1993). However, the observation of relatively low ICAM-1 expression in unstimulated primary corneal epithelial cells in vitro, as against no detectable expression in vivo, is similar to what has been reported for other ocular epithelial cells. For example, when ICAM-1 was examined on the conjunctival epithelium in vivo no constitutive expression was detected (Ciprandi et al., 1993 ; Vorkauf et al., 1995), but low constitutive expression was observed in vitro on a continuously cultured conjunctival epithelial cell line (Canonica et al., 1995). A similar pattern was also seen with cultured retinal pigment epithelial cells (Duguid, Boyd and Mandel, 1992 ; Elner et al., 1992 ; Platts et al., 1995) when compared to intact tissue (Whitcup et al., 1992). It should be noted that, in most studies, the detection of ICAM-1 expression in vivo was performed in tissue sections using immunohistochemical techniques. The ELISA is more sensitive than immunohistochemistry. It is therefore possible that a low level of ICAM-1 expression may be present in vivo, but not be detectable immunohistochemically. Recent reports have shown that pro-inflammatory cytokines augment the expression of ICAM-1 on a variety of ocular cell types in vivo and in vitro. For example, TNF-α, IFN-γ and IL-1β modulate ICAM-1 expression on cultured human corneal endothelial (Elner et al., 1991 ; Pavilack et al., 1992) and stromal cells (Pavilack et al., 1992), and retinal pigment epithelium (Elner et al., 1992 ; Platts et al., 1995). IFN-γ also up-regulates ICAM-1 on the conjunctival epithelium (Bouchard et al., 1996). Using flow cytometry, Iwata et al. (Iwata et al., 1997) have reported the expression of ICAM-1 on HCEs in vitro after stimulation with relatively high concentrations of IFN-γ (1000 U ml−") and TNF-α (2000 µg ml−"). These data are in agreement with our results. Although, it should be noted that the concentrations used by Iwata et al., are " 10 times that needed for maximal ICAM-1 stimulation on HCEs (see Figs 2 and 5 in this report). Our data demonstrate TNF-α and IFN-γ modulation of ICAM-1 expression on cultured human corneal epithelium. IFN-γ was by far the most potent stimulator of ICAM-1 (" 20-fold), while TNF-α elicited a relatively modest but consistent increase in ICAM-1 expression (" 6-fold). It was not surprising that the HCEs expressed an increased amount of ICAM-1 in response to stimulation by IFN-γ, since this cytokine has been shown to up-regulate ICAM-1 on virtually every epithelium tested to date (Bloemen et al., 1993 ; Dustin et al., 1986, 1988 ; Elner et al., 1992 ; Platts et al., 1995 ; Rothlein et al., 1988). The fact that a consistent and significant increase in ICAM-1 expression was detected as early as 6 hr post-stimulation with IFN-γ and TNF-α, argues for a direct effect via IFN-γ and TNF-α receptors on HCEs and not through

C O R N E A L E P I T H E L I A L I C AM-1 E X P R E S S I ON

the induction of another cytokine(s). The fact that ICAM-1 protein was detected at 6 hr and not significantly before also argues for the need for protein synthesis to occur in HCEs, similar to what has been demonstrated in other ICAM-1 expressing cell types (Rothlein et al., 1986). This is also supported by the observed up-regulation of ICAM-1 specific mRNA in IFN-γ treated cells compared to the controls. TNF-α and IFN-γ are known to act synergistically to up-regulate ICAM-1 in a variety of epithelia such as human bronchial epithelial cells (Bloemen et al., 1993) and human keratinocytes (Barker et al., 1990 ; Dustin et al., 1988). However, when HCEs were incubated with a combination of TNF-α and IFN-γ no synergism was detected, counter to what has been reported for other epithelial cell types (Barker et al., 1990 ; Bloemen et al., 1993 ; Dustin et al., 1988). The reason for this difference is not readily apparent at this point, but may reflect a fundamental difference between the corneal and other epithelia. Not all of the pro-inflammatory cytokines examined modulated ICAM-1 expression in the HCEs. The interleukin family tested, IL-1-α and -β and IL-4, did not appear to have any biologically significant effect under the conditions chosen. While a significant effect was detected at 10 ng IL-1α ml−", this effect occurred after 72 hr of stimulation ; the minor ( 2-fold) upregulation in ICAM-1 expression is most likely due to the production of some other cytokine. It was not surprising that IL-4 did not modulate ICAM-1 expression on HCEs since IL-4 is generally a negative immunomodulator of ICAM-1 expression (Schlaak et al., 1995). IL-4 is not known to up-regulate ICAM-1 expression in normal epithelium (Colgan et al., 1994), but only under extraordinary circumstances such as carcinoma (Obiri, Tandon and Puri, 1995). While relatively high levels of IL-4 have been found in the tears of patients with severe ocular allergy compared to their normal counterparts (Fujishima et al., 1995), IL-4 is probably not responsible for the increased ICAM-1 expression observed in these patients. However, IL-4 may affect the corneal epithelium in other ways, such as modulating cytokine release. It is interesting that neither IL-1α nor -β significantly affected ICAM-1 levels in HCEs, since both are known inducers of ICAM-1 on other epithelia (Dustin et al., 1986 ; Patel et al., 1995), including retinal pigment epithelium (Elner et al., 1992 ; Platts et al., 1995) which respond to IL-1β. However, exceptions have been reported. For example, human skin epithelial cells are not responsive to IL-1β (Dustin et al., 1988). The inability of IL-1α or -1β to stimulate ICAM-1 on HCEs was not due to a lack of cytokine biological activity, since the biological activity of IL-1α and -1β was confirmed in A549 cells. We have also determined that HCEs can respond to IL-1α by releasing chemoattractant cytokines (Trocme et al., 1997). These data suggest that HCEs have receptors for IL-1α. However, these receptors appear to propa-

391

gate a cellular signal for chemokine release, but not for ICAM-1 production. ICAM-1 is believed to play a crucial role in eosinophil recruitment, attachment and degranulation during allergic inflammation, and hence may contribute to corneal epithelial disease and ulceration associated with sight-threatening forms of ocular allergy. We have previously proposed a model in which ICAM-1 plays a pivotal role in the pathogenesis of corneal ulceration as seen in severe ocular allergy such as VKC (Gill et al., 1996). The role of ICAM-1 in eosinophil-corneal epithelial cell interactions during ocular disease, such as allergy requires further clarification. Our observations of ICAM-1 up-regulation in cytokine-stimulated HCEs suggest new avenues of investigation regarding the mechanisms of eosinophil infiltration and retention in the corneal epithelium and may provide the basis for the development of new treatment strategies.

Acknowledgements The authors would like to thank Mrs Kim Palkowetz for performing the flow cytometry ; Dr Henry Brysk for critically reading this manuscript ; Dr Todd Pappas for performing the statistical analysis ; and the UTMB Molecular Biology Training Core Facility for use of the image analyser. Supported by grants from the John Sealy and Knights Templar Foundations, and the Galveston Lions Club (JY-B ; SDT) ; Research to Prevent Blindness, Inc., NY, NY, U.S.A. ; National Institutes of Health grants DE08477 (MMB), and DC02129 (JAP) ; and a gift to the Dept. of Ophthalmology and Visual Sciences from Mrs Duff Beverly, Hitchcock, TX, U.S.A.

References Barker, J. N. W. N., Sarma, V., Mitra, R. S., Dixit, V. M. and Nickoloff, B. J. (1990). Marked synergism between tumor necrosis factor-α and interferon-γ in regulation of keratinocyte-derived adhesion molecules and chemotactic factors. J. Clin. Invest. 85, 605–8. Bloemen, P. G. M., Tweel, M. C. v. d., Hendricks, P. A. J., Engels, F., Wagenaar, S. S., Rutten, A. A. J. J. L. and Nijkamp, F. P. (1993). Expression and modulation of adhesion molecules on human bronchial epithelial cells. Amer. J. Respir. Cell Molec. Biol. 9, 586–93. Bouchard, C. S., Lasky, J. B., Cundiff, J. E. and Smith, B. S. (1996). Ocular surface upregulation of intercellular adhesive molecule-1 (ICAM-1) by local interferongamma (IFN-γ) in the rat. Curr. Eye Res. 15, 203–8. Canonica, G. W., Ciprandi, G., Buscaglia, S., Pesce, G. and Bagnasco, M. (1994). Adhesion molecules of allergic inflammation : recent insights into their functional roles. Allergy 49, 135–41. Canonica, G. W., Ciprandi, G., Pesce, G. P., Buscaglia, S., Paolieri, F. and Bagnasco, M. (1995). ICAM-1 on epithelial cells in allergic subjects : A hallmark of allergic inflammation. Internat. Arch. Allergy Immunol. 107, 99–102. Carlos, T. M. and Harlan, J. M. (1994). Leukocyte-endothelial adhesion molecules. Blood 84, 2068–101. Ciprandi, G., Buscaglia, S., Pesce, G., Villaggio, B., Bagnasco,

392

M. and Canonica, G. W. (1993). Allergic subjects express intercellular adhesion molecule-1 (ICAM-1 or CD54) on epithelial cells of conjunctiva after allergen challenge. J. Allergy Clin. Immunol. 91, 783–92. Colgan, S. P., Resnick, M. B., Parkos, C. A., Delp-Archer, C., McGuirk, D., Bacarra, A. E., Weller, P. F. and Madara, J. L. (1994). IL-4 directly modulates function of a model human intestinal epithelium. J. Immunol. 153, 2122–9. Del Prete, G. (1992). Human Th1 and Th2 lymphocytes : their role in the pathophysiology of atopy. Allergy 47, 450–5. Duguid, I. G. M., Boyd, A. W. and Mandel, T. E. (1992). Adhesion molecules are expressed in the human retina and choroid. Curr. Eye Res. 11, 153–9. Dustin, M. L., Rothlein, R., Bhan, A. K., Dinarello, C. A. and Springer, T. A. (1986). Induction by IL-1 and interferon, tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 137, 245–54. Dustin, M. L., Singer, K. H., Tuck, D. T. and Springer, T. A. (1988). Adhesion of T lymphoblasts to epidermal keratinocytes is regulated by interferon γ and is mediated by intercellular adhesion molecule 1 (ICAM1). J. Exp. Med. 167, 1323–40. Elner, V. M., Elner, S. G., Pavilack, M. A., Todd, R. F. III, Yue, B. Y. and Huber, A. R. (1991). Intercellular adhesion molecule-1 in human corneal endothelium. Modulation and function. Amer. J. Pathol. 138, 525–36. Elner, S. G., Elner, V. M., Pavilack, M. A., Todd III, R. F., Mayo-Bond, L., Franklin, W. A., Strieter, R. M., Kunkel, S. L. and Huber, A. R. (1992). Modulation and function of intercellular adhesion molecule-1 (CD54) on human retinal pigment epithelial cells. Lab. Invest. 66, 200–11. Foster, C. S. (1995). The pathophysiology of ocular allergy : Current thinking. Allergy 50, 6–9 ; discussion 34-8. Fujishima, H., Takeuchi, T., Shinozaki, N., Saito, I. and Tsubota, K. (1995). Measurement of IL-4 in tears of patients with seasonal allergic conjunctivities and vernal keratoconjunctivitis. Clin. Exp. Immunol. 102, 395–98. Galli, S. J., Gordon, J. R. and Wershil, B. K. (1993). Mast cell cytokines in allergy and inflammation. Agents and Actions Suppl. 43, 209–20. Galli, S. J. and Wershil, B. K. (1996). Immunology —the two faces of the mast cell. Nature 381, 21–2. Gill, K. S., Yannariello-Brown, J., Patel, J., Nakajima, N., Rajaraman, S. and Trocme, S. D. (1996). ICAM-1 expression in corneal epithelium of a vernal keratoconjunctivitis patient. Cornea 16, 107–11. Goldberg, M. F., Ferguson, T. A. and Pepose, J. S. (1994). Detection of cellular adhesion molecules in inflamed human corneas. Ophthalmol. 101, 161–8. Gosset, P., Tillie-Leblond, I., Janin, A., Marquette, C.-H., Copin, M.-C., Wallaert, B. and Tonnel, A.-B. (1995). Expression of E-selectin, ICAM-1 and VCAM-1 on bronchial biopsies from allergic and non-allergic asthmatic patients. Internat. Arch. Allergy Immunol. 106, 69–77. Harvima, I. T., Horsmanheimo, L., Naukkarinen, A. and Horsmanheimo, M. (1994). Mast cell proteinases and cytokines in skin inflammation. Arch. Dermatol. Res. 287, 61–7. Hobden, J. A., Masinick, S. A., Barrett, R. P. and Hazlett, L. D. (1995). Aged mice fail to upregulate ICAM-1 after Pseudomonas aeruginosa corneal infection. Invest. Ophthalmol. Vis. Sci. 36, 1107–14. Iwata, M., Sawada, S., Sawa, M. and Thoft, R. A. (1997). Mechanisms of lymphocyte adhesion to cultured human corneal epithelial cells. Curr. Eye Res. 16, 751–60. Laemmli, U. K. (1970). Cleavage of structural proteins

J. Y A N N A R I E L LO-B R O W N E T A L.

during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680–5. Leff, A. R., Hamann, K. J. and Wegner, C. D. (1991). Inflammation and cell-cell interactions in airway hyperresponsiveness. Amer. J. Physiol. (Lung Cell. Mol. Physiol. 4) 260, 1189–205. Maggi, E., Biswas, P., Del Prete, G., Parronchi, P., Macchia, D., Simonelli, C., Emmi, L., De Carli, M., Tiri, A., Ricci, M. et al. (1991). Accumulation of Th-2-like helper T cells in the conjunctiva of patients with vernal conjunctivitis. J. Immunol. 146, 1169–74. Nakajima, H., Gleich, G. J. and Kita, H. (1996). Constitutive production of IL-4 and IL-10 and stimulated production of IL-8 by normal peripheral blood eosinophils. J. Immunol. 156, 4859–66. Obiri, N., Tandon, N. and Puri, R. (1995). Up-regulation of intercellular adhesion molecule 1 (ICAM-1) on human reneal cell carcinoma cells by interleukin-4. Internat. J. Cancer 61, 635–42. Paolieri, F., Battifora, M., Riccio, A. M., Pesce, G., Canonica, G. W. and Bagnasco, M. (1997). Intercellular adhesion molecule-1 on cultured human epithelial cell lines : influence of proinflammatory cytokines. Allergy 52, 521–31. Patel, J. A., Kunimoto, M., Sim, T. C., Garofalo, R., Eliott, T., Baron, S., Ruuskanen, O., Chonmaitree, T., Orgra, P. L. and Schmalstieg, F. (1995). Interleukin-1 alpha mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus. Amer. J. Resp. Cell Molec. Biol. 13, 602–9. Pavileck, M. A., Elner, V. M., Elner, S. G., III, R. F. T and Huber, A. R. (1992). Differential expression of human corneal and perilimbal ICAM-1 by inflammatory cytokines. Invest. Ophthalmol. Vis. Sci. 33, 564–73. Philipp, W. (1994). Leukocyte adhesion molecules in rejected corneal allografts. Graefe’s Arch. Clin. Exper. Ophthalmol. 232, 87–95. Platts, K. E., Benson, M. T., Rennie, I. G., Sharrard, R. M. and Rees, R. C. (1995). Cytokine modulation of adhesion molecule expression on human retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 36, 2262–9. Rothlein, R., Dustin, M. L., Marlin, S. D. and Springer, T. A. (1986). A human intercellular adhesion molecule (ICAM-1) is distinct from LFA-1. J. Immunol. 137, 1270–4. Rothlein, R., Czajkowski, M., O’Neill, M. M., Marlin, S. D., Mainolfi, E. and Merluzzi, V. J. (1988). Induction of intercellular adhesion molecule 1 on primary and continuous cell lines by pro-inflammatory cytokines. J. Immunol. 141, 1665–9. Schlaak, J., Schwarting, A., Knolle, P., Meyer zum Buschenfelde, K.-H. and Mayet, W. (1995). Effects of Th1 and Th2 cytokines on cytokine production and ICAM-1 expression on synovial fibroblasts. Ann. Rheumatic Dis. 54, 560–5. Trocme, S. D. and Aldave, T. (1994). The eye and the eosinophil. Surv. Ophthalmol. 39, 241–52. Trocme, S. D., Kephart, G. M., Bourne, W. M., Buckley, R. J. and Gleich, G. J. (1993). Eosinophil granule major basic protein deposition in corneal ulcers associated with vernal keratoconjunctivitis. Am. J. Ophthalmol. 115, 640–3. Trocme, S. D., Kephart, G. M., Allansmith, M. R., Bourne, W. M. and Gleich, G. J. (1989). Conjunctival deposition of eosinophil granule major basic protein in vernal keratoconjunctivitis and contact lens-associated giant papillary conjunctivitis. Am. J. Ophthalmol. 108, 57–63. Trocme, S. D., Hallberg, C. K., Haberle, H., Gill, K. S., Nakajima, M., Ernst, P. and Yannariello-Brown, J.

C O R N E A L E P I T H E L I A L I C AM-1 E X P R E S S I ON

(1997). Release of RANTES from primary human corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. (abstract) 37, S859. Vorkauf, W., Vorkauf, M., Nolle, B. and Duncker, G. (1995). Adhesion molecules in normal and pathological corneas. An immunohistochemical study using monoclonal antibodies. Graefe’s Arch Clin. Exper. Ophthalmol. 233, 209–19. Whitcup, S. M., Chan, C.-C., Li, Q. and Nussenblatt, R. B. (1992). Expression of cell adhesion molecules in posterior uveitis. Archives of Opththalmol. 110, 662–6.

393

Whitcup, S. M., Nussenblatt, R. B., Price, F. W., Jr. and Chan, C. C. (1993). Expression of cell adhesion molecules in corneal graft failure. Cornea 12, 475–80. Whitcup, S. M., Hikita, N., Shirao, M., Miyasaka, M., Tamatani, T., Mochizuki, M., Nussenblatt, R. B. and Chan, C.-C. (1995). Monoclonal antibodies against CD54 (ICAM-1) and CD11a (LFA-1) prevent and inhibit endotoxin-induced uveitis. Exp. Eye Res. 60, 597–601. Yannariello-Brown, J. and Madri, J. A. (1990). Characterization of a 48Kd collagen type IV binding protein from aortic endothelial cells. Biochem. J. 265, 383–92.