May 2003
Volume 44, Issue 5
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Immunology and Microbiology  |   May 2003
Differential and Cooperative Effects of TNFα, IL-1β, and IFNγ on Human Conjunctival Epithelial Cell Receptor Expression and Chemokine Release
Author Affiliations
  • James L. Stahl
    From the Departments of Medicine and
  • Ellen B. Cook
    From the Departments of Medicine and
  • Frank M. Graziano
    From the Departments of Medicine and
  • Neal P. Barney
    Ophthalmology and Visual Sciences, School of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2010-2015. doi:10.1167/iovs.02-0721
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      James L. Stahl, Ellen B. Cook, Frank M. Graziano, Neal P. Barney; Differential and Cooperative Effects of TNFα, IL-1β, and IFNγ on Human Conjunctival Epithelial Cell Receptor Expression and Chemokine Release. Invest. Ophthalmol. Vis. Sci. 2003;44(5):2010-2015. doi: 10.1167/iovs.02-0721.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To gain better understanding of conjunctival epithelial cell responses to proinflammatory cytokines, the individual and combined effects of TNFα, IL-1β, and IFNγ on chemokine release (IL-8, regulated on activation normal T-cell expressed and secreted [RANTES]) and surface receptor expression (intercellular adhesion molecule [ICAM]-1, and HLA-DR, -DP, and -DQ) were examined.

methods. Conjunctival epithelial cells were isolated from cadaveric conjunctival tissues and cultured in 24-well plates until almost confluent. Recombinant cytokines (0.005–50 ng/mL) were added, alone or in various combinations, 24 hours before harvesting of supernates for ELISAs and cells for flow cytometry.

results. TNFα, IL-1β, and IFNγ had distinctive individual and combined effects on the parameters tested. Although TNFα and IL-1β had similar and synergistic effects on increasing expression of ICAM-1, IL-1β was a more potent upregulator of the release of IL-8 than was TNFα. Upregulation of IL-8 was additive when IL-1β was combined with TNFα. Neither TNFα nor IL-1β increased expression of HLA. In contrast, IFNγ was a potent upregulator of both surface receptors (ICAM-1 and HLA) but IFNγ alone had no effect on mediator release (IL-8 and RANTES). Release of RANTES required two cytokine signals, with IFNγ and TNFα being the most potent combination.

conclusions. Knowledge of the differential and combined effects of proinflammatory cytokines on conjunctival epithelial cells allows better understanding of ocular inflammation.

Evidence suggests that conjunctival epithelial cells play an active role in ocular inflammation. For example, in ocular allergic inflammation, histamine receptors expressed on conjunctival epithelial cells represent the major targets for ocular antihistamines 1 and proinflammatory cytokines released from allergen-activated mast cells have been shown to result in activation of epithelial cells. 2 3 Immunologically activated epithelial cells of the ocular surface also play a role in chronic inflammation associated with keratoconjunctivitis sicca (KCS). 4 5 6 7 Activated conjunctival epithelial cells release biologically active compounds that are essential in the pathogenesis of ocular inflammation, including lipid mediators, eicosanoids, growth factors, and a variety of cytokines and chemokines. 8 9 Specifically, cultured conjunctival epithelial cells can be induced to express the chemokines interleukin (IL)-8 (chemotactic for neutrophils and eosinophils) and regulated on activation and normal T-cell expressed and secreted (RANTES, chemotactic for T cells and eosinophils). 9 Furthermore, increased expression of intercellular adhesion molecule (ICAM)-1 and human leukocyte antigen (HLA) on the conjunctival epithelium are considered hallmarks of both ocular allergic inflammation and dry-eye disease. 5 6 10 In vivo studies have indicated that there are specific molecular differences in patterns of conjunctival epithelial cell expression of these chemokines and surface receptors in various diseases of the ocular surface. 4 5 6 9 10  
It has been demonstrated in multiple tissues, including the conjunctival epithelium, that stimulation of epithelial cells through proinflammatory cytokines such as TNFα, IL-1β, and IFNγ results in upregulation of surface receptor expression and mediator release. 2 3 4 5 6 7 8 11 12 There is evidence for participation of these proinflammatory cytokines in the pathogenesis of acute and chronic ocular inflammation. Specifically, TNFα concentrations in tears are increased after allergen provocation and the ratio of TNFα to IFNγ concentrations is increased in tears from allergic subjects compared with nonallergic subjects. 13 14 Both TNFα and IL-1 (α and β) are also increased in the conjunctival epithelium of patients with Sjögren syndrome KCS, and the levels of IL-1 RNA directly correlate with intensity of corneal fluorescein staining and decreased conjunctival goblet cell density. 4 15 This increase in IL-1 is accompanied by a decrease in anti-inflammatory IL-1 receptor antagonist (IL-1ra). In ocular allergic inflammation, IL-1 also appears to play a role, in that IL-1ra suppresses allergic eye disease in a murine model for allergic conjunctivitis. 16 In addition, T cells expressing IFNγ have been reported in atopic keratoconjunctivitis (AKC), vernal keratoconjunctivitis (VKC), and Sjögren syndrome. 17 18 19 20  
Although these studies provide insight, the specific mechanisms for regulation of these processes in conjunctival epithelial cells are not fully understood. Therefore, to investigate further the individual and combined effects of TNFα, IL-1β, and IFNγ on primary cultures of human conjunctival epithelial cells’ release of IL-8 and RANTES and surface expression of ICAM-1 and HLA, the following studies were conducted. 
Methods
Reagents and Solutions
Collagenase (type I), hyaluronidase (type I-S), trypsin-EDTA, HEPES, Triton X-100, trypan blue, a density gradient (Percoll; Sigma Chemical Co., St. Louis, MO), gentamicin, penicillin-streptomycin, amphotericin, Hanks’ balanced salt solution (HBSS; without Ca2+, Mg2+, or phenol red), bovine serum albumin (BSA), NaN3, phenylmethylsulfonyl fluoride, and mouse anti-human pancytokeratin fluorescein isothiocyanate (FITC)–conjugated antibody were obtained from Sigma Chemical Co. Keratinocyte growth medium (KGM) was obtained from Clonetics Corp. (San Diego, CA). Collagen/fibronectin (FNC Coating Mix) was obtained from Biological Research Faculty and Facility (Ijamsville, MD). Mouse anti-human pan HLA-FITC–conjugated antibody (reacts with all major histocompatibility class II HLA-DR and DP antigens and most DQ antigens) was obtained from PharMingen (San Diego, CA), and mouse anti-human ICAM-1 phycoerythrin (PE)–conjugated antibody was obtained from BD Biosciences (San Jose, CA). Appropriate isotype control antibodies were purchased from the same manufacturer as the respective antibodies. Recombinant human IFNγ was obtained from R&D Systems (Minneapolis, MN), recombinant human IL-1β from BioSource International (Camarillo, CA), and recombinant human TNFα from Genzyme Diagnostics (Cambridge, MA). The IL-8 and RANTES ELISAs were obtained from BioSource International, Inc. 
The Tyrode physiological salt solution plus gelatin (TG) used in these studies consisted of (mM) NaCl, 137; KCl, 2.6; NaH2PO4, 0.35; NaHCO3, 11.9; glucose, 5.5; and gelatin 1 g/L, adjusted to pH 7.4 with HCl. TGCM is TG with added CaCl2 (2 mM) and MgCl2 (1 mM). The density gradient (Percoll; Sigma Chemical Co.) stock solution was prepared by mixing the commercial solution and 10× HEPES buffer plus dH2O to obtain an osmolality of 285 mOsm/kg H2O. The desired density of the gradient was prepared by mixing the stock solution with TG. HBSS-BAP, used as a flow cytometry staining buffer, consisted of HBSS, BSA (1 g/L), NaN3 (0.5 g/L), and phenylmethylsulfonyl fluoride (18 mg/L in 4 mL ethanol). 
Epithelial Cell Isolation, Purification, and Culture
Modifications of previously reported methods for obtaining purified conjunctival epithelial cells were used in these studies. 21 Briefly, human conjunctival tissue was obtained from organ-tissue donors (8–10 sets of tissue per experiment obtained through the Lion’s Eye Bank of Wisconsin, a nationwide network of eye banks and the National Disease Research Interchange). Upper and lower bulbar conjunctivae aseptically collected within 8 hours after death (average time, 4.5 hours) were transported in corneal preservation medium (Dexsol; Chiron Ophthalmics, Irvine, CA) and stored at 4°C for up to 5 days. Eight to 10 sets of tissue weighing 4 to 5 g were used per experiment. Hyaluronidase and collagenase were used to digest tissue. The digestion process (30 minutes at 37°C on a rotating shaker) was first performed at a low concentration of enzymes (two digests at 200 U/g in a 10-mL final volume). This was followed by tissue digestion at a high concentration of enzymes (three to six digests at 2000 U/g in a 10-mL final volume). Each digest was followed by washing of the enzyme-treated tissue (with TGCM) over a 100-μm nylon mesh filter to collect freed cells. After the digestion procedure, the freed cells were pelleted, pooled, resuspended in TG, and layered over a single-density gradient (1.041 g/mL; Percoll; Sigma Chemical Co.) and centrifuged (500g, 20 minutes). The top cell layer (epithelial cells) was harvested, washed, and resuspended in KGM (without hydrocortisone, at a concentration of 1 × 106 cells/mL) and transferred to collagen/fibronectin-coated 24-well plates (0.5 mL/well) for culture at 37°C. Media were changed every 48 hours until confluence. Purity was determined by flow cytometric analysis of mouse anti-human pancytokeratin-FITC antibody staining of fixed and permeabilized cells, as previously reported. 21  
Treatment of Conjunctival Epithelial Cell Monolayers
Conjunctival epithelial cells (one to two passages) were cultured on 24-well plates until almost confluent (24–48 hours after passage). Recombinant TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) were added to the cultures, alone or in various (all) combinations for 24 hours at 37°C. After 24 hours, the supernates were collected and stored at −70°C until analysis. 
Flow Cytometric Analysis of Surface Receptors on Conjunctival Epithelial Cells
After the supernatants were removed, the epithelial cell monolayers were harvested with trypsin-EDTA and resuspended in 220 μL HBSS-BAP (20 μL removed for cell counts and 200 μL used for staining). Two-color staining was used to measure surface HLA (HLA-DR, -DP, and -DQ) and ICAM-1 expression. Each tube of 100 μL of viable cells (not fixed, 3.0–5.0 × 104 cells/tube) were stained with mouse anti-human pan-HLA-FITC–conjugated antibody and mouse anti-human ICAM-1-PE–conjugated antibody or mouse IgG2a-PE–conjugated antibody isotype control and mouse IgG2a-FITC–conjugated antibody isotype control, using the manufacturer’s recommended amounts (10 μL/tube). The stained cells were incubated on ice for 30 minutes, washed and resuspended in 300 μL/tube of HBSS-BAP for analysis. Propidium iodide was added to each tube to determine viability. Data were acquired with a flow cytometer (FACScan; BD Biosciences) operated with the accompanying software (CellQuest; BD Biosciences). The data, based on gating of viable cells only (10,000 viable cells counted/tube), was analyzed geometrically (WinMDI; http://www.facs.scripps.edu; the Scripps Research Institute, La Jolla, CA). Data are reported as percentage of positive-staining cells. 
Measurement of IL-8 and RANTES Release from Conjunctival Epithelial Cells
The levels of IL-8 and RANTES in the epithelial cell supernates were determined by commercial ELISA according to the manufacturers’ instructions. Cell counts were performed on the cells harvested as above for flow cytometric analysis (Coulter Counter; Model ZM; Beckman Coulter Corp., Miami, FL) to determine the number of epithelial cells per milliliter. Data are reported as the concentration of cytokines per million cells. 
Statistical Analyses
Data were computer analyzed (SAS software; SAS Institute, Cary, NC). A general linear-model analysis of variance (ANOVA) with preplanned comparisons was computed to generate two-tailed probabilities. The Fisher least-significant-difference test was used to make appropriate post-ANOVA comparisons. P ≤ 0.05 was considered statistically significant. Potential cytokine interactions were evaluated by comparing the calculated additivity (response to cytokine x alone + response to cytokine y alone) with the experimental additivity (response to simultaneous stimulation with cytokine x and cytokine y) for each combination of concentrations tested. Interactions of cytokines were defined as follows 22 : additive when the calculated additivity was not significantly different from the experimental additivity; nonadditive when the experimental additivity was significantly less than the calculated additivity; or synergistic when the experimental additivity was significantly greater than the calculated additivity. All data are presented as the mean ± SEM of results from four to seven separate experiments. 
Results
ICAM-1 and HLA
Conjunctival epithelial cells expressed ICAM-1 constitutively. Figure 1A shows a representative overlay histogram of ICAM-1 staining of conjunctival epithelial cells stimulated with 5 ng/mL of each cytokine separately, compared with unstimulated cells. All three recombinant cytokines alone upregulated ICAM-1 expression in a concentration-dependent manner; however, expression was two times greater with IFNγ treatment (up to 90% positive cells) compared with IL-1β or TNFα treatment (up to 45% positive cells for both; Fig. 1B ). When TNFα and IL-1β were combined, ICAM-1 expression was increased over that induced by either cytokine alone at 5 and 50 ng/mL (all combinations were significantly different compared with either cytokine alone); however, the effect was subadditive. This is illustrated in the bar graph in Figure 1C , which shows the individual and combined effect of stimulation with 5 ng/mL TNFα and IL-1β on expression of ICAM-1, compared with the calculated combined effect. The experimental combined effect was significantly less than the calculated combined effect (see the Methods section). A similar effect was observed at other dose combinations (data not shown). When either IL-1β or TNFα was combined with IFNγ treatment, no effect on ICAM-1 expression was observed compared with IFNγ treatment alone. 
HLA was not expressed constitutively on conjunctival epithelial cells. Figure 2A shows a representative overlay histogram of HLA staining of conjunctival epithelial cells stimulated with 5 ng/mL of each cytokine separately, compared with unstimulated cells. HLA expression was stimulated by IFNγ treatment in a concentration-dependent manner, but not by TNFα or IL-1β treatment (Fig. 2B) . No synergistic or additive effects on conjunctival epithelial cell expression of HLA were observed with any combination of cytokines. It should also be noted that at the 50 ng/mL concentration of IFNγ, some loss of viability (10%–15%) was observed, by using propidium iodide uptake. 
IL-8 and RANTES
IL-8 was released constitutively from conjunctival epithelial cells. Release of IL-8 was upregulated by IL-1β and to a lesser extent by TNFα (Fig. 3A) . IFNγ treatment alone did not increase the IL-8 level above that of constitutive release. An effect of IFNγ on IL-8 release (increased) was observed only when IFNγ (0.5 and 5.0 ng/mL) was combined with IL-1β (50.0 ng/mL; data not shown). TNFα and IL-1β together had an additive effect on release of IL-8 at all combinations of doses. The bar graph in Figure 3B is representative of the additive effect on release of IL-8 observed at 5 ng/mL IL-1β and TNFα. In Figure 3B , the individual and combined effect of stimulation with 5 ng/mL TNFα and IL-1β on IL-8 release is compared with the calculated combined effect, which is the same as the experimental combined effect (see the Methods section). A similar effect was observed at other dose combinations (data not shown). 
In contrast to IL-8, RANTES was not released constitutively from conjunctival epithelial cells, and RANTES release was not significantly stimulated by any cytokine alone. However, the combination of any two cytokines stimulated release of RANTES, with IFNγ and either TNFα or IL-1β being the most potent combinations. Figure 4 illustrates the effect of TNFα and IFNγ together on release of RANTES (IL-1β and IFNγ together produced equivalent results). Between TNFα and IFNγ and between IL-1β and IFNγ, there was a trend toward a synergistic interaction on release of RANTES that did not reach statistical significance. It is also important to note that IL-8 was released at much greater concentrations than RANTES (3–1100 ng/million cells versus 0 to 18 pg/million cells, respectively). 
Discussion
This study demonstrates that stimulation of conjunctival epithelial cells with TNFα, IL-1β, and IFNγ have individual and combined effects on conjunctival epithelial cells in vitro. It is well known that TNFα and IL-1β promote activation of some of the same genes that modulate inflammation. In these studies, we observed that, although treatment with TNFα and IL-1β resulted in similar upregulation of ICAM-1 expression, IL-1β was a more effective potentiator of IL-8 release. Furthermore, when TNFα and IL-1β were applied in combination, the effect was additive for release of IL-8. Cooperation between proinflammatory cytokines has been well documented in the literature. Possible mechanisms include signaling through separate pathways leading to coordinated binding of multiple transcription factors to the IL-8 gene and/or posttranscriptional regulation; however, the latter mechanism is not commonly reported in cooperative interactions between cytokines. 23 Although upstream signaling from their respective receptors is distinctly different, TNFα and IL-1β overlap in activation of downstream transcription factors. In the case of the IL-8 gene, transcriptional regulation by TNFα and IL-1β can involve multiple transcription factors. In some cell types, including epithelial cell lines, TNFα signaling primarily involves activation of nuclear factor (NF)-κB and activator protein (AP)-1, whereas IL-1β primarily activates NF-κB and nuclear factor (NF)-IL6. 24 NF-κB binding to the promoter appears to be essential for IL-8 gene activation, but it must be combined with binding of either AP-1 or NF-IL6. 24 25 The literature suggests that NF-IL6 potently enhances IL-8 promoter activity through a physical interaction with NF-κB at the promoter. 26 Consequently, it is not surprising that IL-1β is a more potent stimulator of the release of IL-8 (because it activates NF-IL6) and that simultaneous stimulation with TNFα and IL-1β would result in enhanced release. 
Combined stimulation of conjunctival epithelial cells with TNFα and IL-1β also enhanced expression of ICAM-1, but the net effect was subadditive. This implies that the pathway to upregulation of ICAM-1 by these two cytokines is shared or redundant, which has been demonstrated to be the case in other cell types. The major signal transduction pathway in activation of the ICAM-1 promoter for both TNFα and IL-1β converges at the level of NF-κB activation, which is sufficient for activation of expression of the ICAM-1 gene. 27  
In contrast, IFNγ was found to be ineffective in promoting release of IL-8 from conjunctival epithelial cells, but was the most potent upregulator of surface receptor expression. In fact, IFNγ was the only cytokine effective in upregulation of HLA. Upregulation of ICAM-1 and HLA by IFNγ primarily occurs through a pathway that is not dependent on NF-κB. IFNγ-responsive genes are activated through both IFN regulatory factor (IRF)-1 and signal transducer and activator of transcription (STAT)-1 transcription factors, which have not been shown to play important roles in activation of the IL-8 gene. 24 26 In our model, we did not observe a synergistic or additive effect of cytokine combinations on HLA protein expression over 24 hours. Although some studies have shown that TNFα is synergistic with IFNγ for upregulation of MHC class II gene expression in some cell types, the mechanisms vary. 28 29 30 However, there is consensus that although TNFα modulates MHC class II gene expression, TNFα alone is not sufficient to activate the class II transactivator, an IFNγ-responsive gene that is the master regulator of MHC class II molecules. 28 29 30 31  
Our data demonstrate a cooperative effect of inflammatory cytokines in induction of the release of RANTES from conjunctival epithelial cells. Various studies have demonstrated that both individual and combined stimulation with IFNγ, TNFα, and/or IL-1β can induce release of RANTES from a variety of cell types. 32 33 34 However, in our study, none of the three cytokines (IFNγ, TNFα, or IL-1β) when added individually was effective in promoting expression of RANTES, whereas any combination of cytokines resulted in some release (with IFNγ combined with either TNFα or IL-1β being the most potent combination). Expression of human RANTES mRNA is differentially regulated based on stimulus and cell type. Furthermore, in some models, expression of RANTES is delayed, whereas, in others, it can be immediate and transient. Given the low concentrations of RANTES protein released in conjunctival epithelial cell supernatants (picograms per 106 cell range, in contrast to nanograms per 106 for IL-8), it is possible that the individual cytokines stimulate expression of RANTES, but the concentration of protein released in 24 hours is too low to detect, or that activation of the RANTES gene is delayed. Enhanced activation of the RANTES gene by combinations of cytokines has been reported. 32 34 For example, activation of the RANTES gene is enhanced by a combination of TNFα and IFNγ due to a cooperative effect of activation of both NF-κB and IRF-1. 32  
Integrating these in vitro findings with information gained from in vivo studies and our knowledge of the biological activities of the components measured has the potential to contribute significantly to our understanding of the pathogenesis of ocular surface inflammation. Discerning the cellular sources of cytokines and modes of activation of these cells is also critical to determining pharmacological targets. In allergic eye disease, the inflammatory cytokines TNFα, IL-1β, and IFNγ are present, yet their cellular sources have not been specifically identified. 12 13 14 15 16 17 During acute ocular allergic disease, conjunctival mast cells are probably the initial source of the proinflammatory cytokines TNFα and IL-1β. We have demonstrated that anti-IgE antibody challenge of purified human conjunctival mast cells results in TNFα release in a concentration-dependent manner. 35 Supernatants from anti-IgE–activated conjunctival mast cells upregulate conjunctival epithelial cell ICAM-1 expression and release of IL-8. 2 3 Furthermore, the mechanism for mast cell supernatant–mediated upregulation of ICAM-1 was determined to be TNFα specific. This was demonstrated in studies using blocking antibody to TNFα and inhibition by the mast cell stabilizer, olopatadine, which also inhibits release of histamine, tryptase, and prostaglandin D2 (PGD2) from conjunctival mast cells. 2 36 Mast cells are also believed to be an important source of IL-1β, and preliminary data (message expression and intracellular protein immunostaining) from our laboratory with conjunctival mast cells support this (Stahl et al., unpublished data, 2002). Therefore, in ocular allergic inflammation, simultaneous release of TNFα and IL-1β from mast cells could explain the increase in IL-8 that has been reported in epithelial cells on the ocular surface in AKC and VKC. 9 In VKC, IL-8 levels in supernates obtained from the ocular surface correlate with numbers of infiltrating eosinophils and neutrophils and severity of corneal lesions. 37  
As mentioned, TNFα, IL-1 (α and β), and IL-8 are increased in the conjunctival epithelium of patients with Sjögren syndrome KCS. Several studies have suggested that the lacrimal gland acinar cell is an important source of IL-1β, which is also increased in tear fluid in dry-eye disease. 15 Furthermore, the levels of IL-1 and IL-8 RNA directly correlate with indicators of disease severity. 4 15 Our studies indicate that this may be in part to because IL-1β is a potent stimulator of the release of IL-8 from conjunctival epithelial cells. 
Although it has been reported that mast cells can release IFNγ, to date we have not confirmed this with conjunctival mast cells. In chronic ocular surface inflammation, T cells are a likely major source of IFNγ. 16 17 As previously mentioned, T cells expressing IFNγ have been reported in AKC, VKC, and Sjögren syndrome. 17 18 19 20 IFNγ is critical for upregulation of surface receptors required for cell-to-cell interactions including migration and antigen presentation. In fact, the antigen-presenting potential of human conjunctival epithelium has been suggested by the concomitant upregulation of CD40 (coreceptor for T-cell activation and antigen presentation) and HLA in vitro. 38 Antigen-presenting capability of corneal epithelial cells and lacrimal acinar cells has been demonstrated. 39 40 Additional studies suggest a role for signaling through ICAM/leukocyte function-associated antigen (LFA)-1 interactions in costimulation of T cells. 41 In vitro it has been shown that retinal pigment epithelial cells are capable of presenting bacterial superantigens to T cells through an interaction involving ICAM-1. 42 These interactions between epithelial cells and IFNγ-secreting T cells may be important components in the maintenance of chronic ocular inflammation. Furthermore, the results presented herein suggest the combination of IFNγ with either TNFα or IL-1β in vivo would result in release of RANTES, potentially explaining the persistent T-cell infiltration that is characteristic of ocular inflammatory conditions that become chronic, such as AKC and Sjögren syndrome KCS. In addition, it has been hypothesized that IFNγ contributes to inhibition of mucus production, as has been shown in airway inflammation. 43  
These studies demonstrate the importance of individual and combined effects of cytokines present during ocular inflammatory processes on conjunctival epithelial cell activation. Selective changes in the balance of proinflammatory mediators, as the result of treatment, could have potential importance in managing ocular inflammation. 
 
Figure 1.
 
Conjunctival epithelial cell monolayers were incubated with recombinant TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for ICAM-1 was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells. (C) Individual and combined effects of stimulation with 5 ng/mL TNFα and IL-1β on expression of ICAM-1, compared with the calculated combined effect.
Figure 1.
 
Conjunctival epithelial cell monolayers were incubated with recombinant TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for ICAM-1 was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells. (C) Individual and combined effects of stimulation with 5 ng/mL TNFα and IL-1β on expression of ICAM-1, compared with the calculated combined effect.
Figure 2.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for pan-HLA was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells.
Figure 2.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for pan-HLA was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells.
Figure 3.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed by ELISA. Data are reported as the concentration of IL-8 per million cells (n = 5–7). (A) Release of IL-8 from conjunctival epithelial cells incubated with TNFα, IL-1β, or IFNγ. (♦) Treatments that were significantly different (P ≤ 0.05) compared with unstimulated cells. (B) Individual and combined effect of stimulation with 5 ng/mL IL-1β and TNFα on expression of IL-8 compared with the calculated combined effect. Although all the data combining IL-1β and TNFα at the full range of doses are not shown, these results are representative of the additive effect observed at various doses of IL-1β and TNFα combined.
Figure 3.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed by ELISA. Data are reported as the concentration of IL-8 per million cells (n = 5–7). (A) Release of IL-8 from conjunctival epithelial cells incubated with TNFα, IL-1β, or IFNγ. (♦) Treatments that were significantly different (P ≤ 0.05) compared with unstimulated cells. (B) Individual and combined effect of stimulation with 5 ng/mL IL-1β and TNFα on expression of IL-8 compared with the calculated combined effect. Although all the data combining IL-1β and TNFα at the full range of doses are not shown, these results are representative of the additive effect observed at various doses of IL-1β and TNFα combined.
Figure 4.
 
RANTES release from conjunctival epithelial cell monolayers incubated with combinations of TNFα and IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed with ELISA. Data are reported as concentration of RANTES per million cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05), compared with unstimulated cells and cells treated with either TNFα or IFNγ alone at the doses indicated.
Figure 4.
 
RANTES release from conjunctival epithelial cell monolayers incubated with combinations of TNFα and IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed with ELISA. Data are reported as concentration of RANTES per million cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05), compared with unstimulated cells and cells treated with either TNFα or IFNγ alone at the doses indicated.
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Figure 1.
 
Conjunctival epithelial cell monolayers were incubated with recombinant TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for ICAM-1 was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells. (C) Individual and combined effects of stimulation with 5 ng/mL TNFα and IL-1β on expression of ICAM-1, compared with the calculated combined effect.
Figure 1.
 
Conjunctival epithelial cell monolayers were incubated with recombinant TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for ICAM-1 was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells. (C) Individual and combined effects of stimulation with 5 ng/mL TNFα and IL-1β on expression of ICAM-1, compared with the calculated combined effect.
Figure 2.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for pan-HLA was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells.
Figure 2.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, or IFNγ (0.005–50 ng/mL) for 24 hours. Staining for pan-HLA was examined by flow cytometry. (A) Representative overlay histograms of ICAM-1 staining. TNFα and IL-1β histograms overlap. (B) Data are reported as the percentage of positively stained cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05) compared with unstimulated cells.
Figure 3.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed by ELISA. Data are reported as the concentration of IL-8 per million cells (n = 5–7). (A) Release of IL-8 from conjunctival epithelial cells incubated with TNFα, IL-1β, or IFNγ. (♦) Treatments that were significantly different (P ≤ 0.05) compared with unstimulated cells. (B) Individual and combined effect of stimulation with 5 ng/mL IL-1β and TNFα on expression of IL-8 compared with the calculated combined effect. Although all the data combining IL-1β and TNFα at the full range of doses are not shown, these results are representative of the additive effect observed at various doses of IL-1β and TNFα combined.
Figure 3.
 
Conjunctival epithelial cell monolayers were incubated with TNFα, IL-1β, and/or IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed by ELISA. Data are reported as the concentration of IL-8 per million cells (n = 5–7). (A) Release of IL-8 from conjunctival epithelial cells incubated with TNFα, IL-1β, or IFNγ. (♦) Treatments that were significantly different (P ≤ 0.05) compared with unstimulated cells. (B) Individual and combined effect of stimulation with 5 ng/mL IL-1β and TNFα on expression of IL-8 compared with the calculated combined effect. Although all the data combining IL-1β and TNFα at the full range of doses are not shown, these results are representative of the additive effect observed at various doses of IL-1β and TNFα combined.
Figure 4.
 
RANTES release from conjunctival epithelial cell monolayers incubated with combinations of TNFα and IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed with ELISA. Data are reported as concentration of RANTES per million cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05), compared with unstimulated cells and cells treated with either TNFα or IFNγ alone at the doses indicated.
Figure 4.
 
RANTES release from conjunctival epithelial cell monolayers incubated with combinations of TNFα and IFNγ (0.005–50 ng/mL) for 24 hours. Supernatants were collected and analyzed with ELISA. Data are reported as concentration of RANTES per million cells (n = 5–7). (♦) Treatments that produced significantly different activity (P ≤ 0.05), compared with unstimulated cells and cells treated with either TNFα or IFNγ alone at the doses indicated.
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