Proinflammatory Chemokine Induction in Keratocytes and Inflammatory Cell Infiltration into the Cornea

Abstract

purpose. To determine the effect of interleukin (IL)-1α and tumor necrosis factor (TNF)-α on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma.

methods. Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1α and TNFα. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte–macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining.

results. IL-1α or TNFα upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma.

conclusions. Proinflammatory chemokine induction in keratocytes is mediated by IL-1α and TNFα. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.

Cytokine-mediated cellular communications are thought to be of paramount importance in development, homeostasis, and wound healing in many organs in which connective and epithelial tissues are conjoined structurally and functionally. The cornea is an exceptional model for investigation of these communications because of the absence of blood vessels, sebaceous glands, hair follicles, or other potentially confounding tissues found in organs such as skin. ^{1 2 3}  

Although there are a number of cytokine receptor systems that may participate in responses associated with corneal injury, many investigators have suggested that the interleukin (IL)-1 system is especially important. IL-1 is produced constitutively in the epithelium, but not in keratocytes, in the unwounded cornea and is released by injury or death of epithelial cells. ^{4 5} IL-1 has been shown to modulate keratocyte apoptosis, ^{6 7} keratocyte-myofibroblast hepatocyte growth factor and keratinocyte growth factor production associated with control of epithelial healing, ^{5 8} and keratocyte-myofibroblast collagenase and metalloproteinase production involved in stromal remodeling. ^{9 10} IL-1 may also modulate the functions of other cytokines, such as the effect of platelet-derived growth factor (PDGF) on stromal fibroblast chemotaxis. ¹¹ Some of these functions of IL-1 appear to be shared by the tumor necrosis factor (TNF)-α TNF receptor system. ¹²  

In this study, gene array technology was used to further explore the effects of IL-1α and TNFα on the expression of cytokines, chemokines, and receptors in the cornea. These experiments confirmed that IL-1α and TNFα upregulate several chemokines in corneal fibroblasts that are involved in chemotaxis and activation of immune cells. Corneal epithelial injury or microinjection of these chemokines attracts inflammatory cells into the corneal stroma. 

Materials and Methods

Cell Culture and Cytokine Treatment

Primary human stromal fibroblasts (HSFs) were cultured from normal donor corneas using the explant method. ⁶ First-passage HSF cells were transferred to complete Eagle’s modified essential medium ⁶ with 0.5% fetal bovine serum and were treated for 6 hours with 20 ng/ml human IL-1α (R&D Systems, Minneapolis, MN), 20 ng/ml human TNFα (R&D Systems), or vehicle. Cells were used immediately after incubation with cytokines or vehicle for isolation of RNA for gene array analysis or RNase protection assay. 

Human Cytokine–Chemokine Receptor Gene Array

An Atlas human cDNA expression array (Clontech, Palo Alto, CA) that has 268 known cytokine–chemokine receptor genes represented was used in these experiments. A complete list of the 268 genes included in this human array can be found at http://www.clontech.com. 

Total RNA was isolated using RNA extraction reagent (TRIzol; Gibco BRL, Rockville, MD) according to the manufacturer’s instructions. After DNase treatment, ³²P-labeled cDNA probe was synthesized from total RNA according to the manufacturer’s protocol. Each cDNA probe was purified using Chroma-spin columns (Clontech). Incorporation of label was assessed using scintillation counting. Equal counts per minute (cpm; 2 × 10⁶) of cDNA probe from the IL- 1α, TNFα, or control group were hybridized in hybridization solution (Express Hyb; Clontech) to the cytokine–chemokine receptor array membranes overnight at 68°C with continuous agitation. The arrays were washed in wash solution 1 (2× SSC, 1% SDS) and wash solution 2 (0.1× SSC, 0.5% SDS) at 68°C. The array membranes were exposed to x-ray film (BioMax MS; Eastman Kodak, Rochester, NY) at −70°C. 

Autoradiographic results were analyzed and compared by computer (Atlas Image 1.1 software; Clontech). This software automatically averages the two hybridization signals for a particular gene and compares this intensity to the same gene in the control array. Exposure times between 4 and 48 hours were used. Lower exposure times were used for comparison of genes with high expression. Results were consistent when different exposure times were compared, but signal spread necessitated analysis of lower exposures for some genes. Using the quantitation obtained with this software for analysis of differences between experimental and control arrays, only positive or negative intensity differences between the cytokine-treated cell–probed arrays and the vehicle-treated cell–probed arrays that were greater than 12,000 were accepted as significant. This cutoff determined by the computer program is arbitrary, but represents a highly significant difference. Further description of this quantitation can be obtained at the Web site provided by the manufacturer (http://www.clontech.com). 

RNase Protection Assay Analysis of MCAF mRNA Production in Human Stromal Fibroblasts

Total RNA was extracted from HSF using extraction reagent (TRIzol; Gibco). A cDNA fragment for human MCAF was generated using polymerase chain reaction (PCR) with primers (5′-TGCTCATAGCAGCCACCTTC-3′ and 5′-TGGGTTTGCTTGTCCAGG-3′) designed according to the published MCAF sequence (GenBank accession number, X14768). The conditions for PCR were 94°C for 1 minute; 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and 72°C for 7 minutes in a programmable thermal controller (PTC-100; MJ Research, Inc., Watertown, MA). The PCR product was cloned into the pCR II vector (Invitrogen, San Diego, CA) and sequenced using standard methods to confirm that the sequence was correct. 

An RNase protection assay (RPA) was performed using the published method. ¹³ Briefly, the RNA probe was synthesized using the linearized MCAF cDNA (253 bp) as a template, along with T7 RNA polymerase (Roche, Indianapolis, IN), [α-³²P]-UTP (800 Ci/mmol; NEN, Boston, MA), and unlabeled nucleotides. ¹³ The RNA probe was purified by ethanol precipitation to remove unincorporated labeled nucleoside triphosphates (Promega, Madison, WI). Twenty micrograms of total cellular RNA ¹³ made from the HSF cells treated with TNFα, IL-1α, or vehicle control were incubated in hybridization buffer containing 2 × 10⁶ cpm of labeled RNA probe at 45°C overnight. The hybridization mixture was digested using 40μ g/ml RNase A and 1000 U/ml RNase T1 (Sigma, St. Louis, MO) at 30°C for 1 hour. The protected MCAF mRNAs were analyzed on a 6% sequencing gel. The dried gel was exposed to film (BioMax; Eastman Kodak) for 2 hours. 

Western Blot Detection of MCAF Protein Production by Stromal Fibroblasts

Confluent first-passage HSFs in 60-mm² culture plates with 1 ml serum-free medium were treated with 20 ng/ml IL-1α (R&D Systems), 20 ng/ml TNFα (R&D Systems), 250 ng/ml Fas-activating IgM (Upstate Biotechnology, Lake Placid, NY), or vehicle for 24 hours or 48 hours. Protein in the conditioned medium was measured with protein detection reagent (Bio-Rad, Richmond, CA) and diluted to a concentration of 0.26 mg/ml with PBS. Twenty-five microliters of diluted conditioned medium was loaded in each lane. Western blot analysis was performed to detect proteins using a published method. ¹³ Two micrograms per milliliter anti-human MCAF (MCP-1) monoclonal antibody (mouse IgG1, no. 23007-111; R&D Systems) were used for the Western blot analysis. The secondary antibody (anti-mouse IgG conjugated with alkaline phosphatase; Promega) and 5-bromo-4-chloro-3-indoyl phosphate 4-toluidine salt/nitroblue tetrazolium chloride (BCIP/NBT) color development substrate (Promega) were used to detect the first antibody according to the instructions of the manufacturer. Prestained protein molecular weight standards (Amersham Life Sciences, Inc., Arlington Heights, IL) were run simultaneously on the 15% polyacrylamide gel to determine the size of the proteins. All other reagents were obtained from Sigma. 

Detection of MCAF Protein by Immunocytochemistry

The rabbit corneoscleral rims were removed with 0.12-mm forceps and sharp Westcott scissors. The tissue was fixed in 4% paraformaldehyde (no. 19202; Electron Microscope Sciences, Fort Washington, PA) at room temperature for 6 hours. After fixation, the corneas were placed in 30% sucrose overnight at 4°C and mounted in optimal temperature cutting (OCT) compound (Tissue-Tek, Torrance, CA) the next day. Cryostat sections were cut at 10 μm thickness and mounted on microscope slides (no. 48311-703; VWR, West Chester, PA). 

The rabbit corneal sections were pretreated with blocking solution containing 5% horse serum provided in an immunostaining kit (Vectastain TM Elite Kit, no. PK-6102; Vector Laboratories, Burlingame, CA), 1% BSA, and 1% Triton-100 (no. T9284; Sigma) in phosphate-buffered saline (PBS; 0.05 M phosphate buffer, 0.137 M NaCl[ pH 7.45]; Sigma) for 30 minutes at room temperature. The mouse monoclonal IgG1 anti-human MCAF antibody, which had been shown by the manufacturer to bind rabbit MCAF, was diluted to 1 μg/ml in PBS containing 1% Triton X-100. The sections were incubated with primary antibody overnight at 25°C. Negative controls were incubated with isotype-matched nonspecific control antibody or without primary antibody. Immunostaining was performed with the kit (Vectastain TM Elite Kit; Vector), according to the manufacturer’s instructions. 3,3-Diaminobenzidine (DAB; no. D-5905; Sigma) was used to visualize the label. Glycerol was applied to the sections on the slide, and a coverslip was placed into position. 

Immunohistochemistry for Monocytes–Macrophages

Corneal epithelial scrape injury was performed with a scalpel blade in one eye of New Zealand White rabbits, by using previously reported methods. ¹⁴ The corneoscleral rims were removed with 0.12-mm forceps and sharp Westcott scissors. The tissue was fixed in 4% paraformaldehyde at room temperature for 6 hours. After fixation, the corneas were placed in 30% sucrose overnight at 4°C and mounted in OCT compound the next day. Cryostat sections were cut at 10-μm thickness and mounted on microscope slides. 

The 24-hour scraped and normal rabbit cornea sections were pretreated with blocking solution containing 5% rabbit serum provided in the staining kit (Vectastain; Vector) and 1% Triton X-100 (no. T9284; Sigma) in PBS for 30 minutes at room temperature. The rat anti-mouse monocyte-macrophage–specific monoclonal antibody (no. RDI-T2008X; R&D Systems Inc.) shown to recognize rabbit antigen was diluted to 1μ g/ml in PBS containing 1% Triton X-100. The sections were incubated with primary antibody overnight at 25°C. Immunolabeling was performed according to the manufacturer’s method. DAB was used to visualize the labeling, and the sections were coverslipped with glycerol. 

Microinjection of MCAF or G-CSF into the Corneal Stroma

Mice (C-56/J6) were used because rabbit chemokines are not available from commercial suppliers. Mice were anesthetized by intraperitoneal injection of xylazine 5 mg/kg and ketamine 50 mg/kg. The eye was anesthetized by topical application of a drop of proparacaine 1% just before microinjection. 

Microinjection was performed using a syringe microinjection system (Hamilton, Reno, NV) with drawn glass capillary needle attached to the blunt needle provided for the system with polyethylene tubing. Mouse MCAF (MCP-1, no. 479-JE-050; R&D Systems, Inc.) or mouse G-CSF (no. 414-CS-025; R& D Systems) were diluted in the carrier solution (1× PBS, 0.5% BSA) at a concentration of 100 ng/μl. The MCAF solution, G-CSF solution, or carrier control solution was drawn into the glass needle. The tip of the sterile needle was passed through the limbus and advanced into the central stroma under direct, magnified observation with an operating microscope. Microinjection of 1 μl was performed, and the needle was withdrawn. After 24 or 48 hours the animal was placed under general anesthesia and killed with 100 mg/kg intraperitoneal pentobarbital. The eyes were removed with fine scissors and forceps, fixed overnight in 37% buffered formaldehyde solution (Sigma), and embedded in paraffin. Eight-micrometer sections were cut with a keratome and placed on 25 × 75 × 1-mm microscope slides (Superfrost Plus; Fisher, Houston, TX). Sections were deparaffinized using standard techniques before hematoxylin and eosin staining. Micrographs were obtained with a fluorescence light microscope (model E600; Nikon, Melville, NY). 

Results

Twenty-eight of the tested cytokine or receptor genes were upregulated in IL-1α–treated HSFs (Fig. 1 , Table 1 ). At least 7 of these genes code for cytokines or chemokines involved in the chemotaxis or activation of inflammatory cells (Table 1) . Twenty genes were downregulated in HSFs in response to IL-1α (Fig. 1 , Table 2 ). Fifteen of the tested genes were upregulated in HSFs in response to TNFα (Fig. 1 , Table 3 ). Thirteen genes were downregulated in response to TNFα (Fig. 1 , Table 4 ). 

There were some genes that were upregulated in response to both IL-1α and TNFα. For example, G-CSF, neutrophil-activating peptide, and erythroid differentiation protein mRNAs were upregulated in HSFs in response to IL-1α or TNFα. 

The gene array experiments with IL-1α and TNFα were performed twice with highly consistent results in the two experiments. 

The upregulation of several genes that code for inflammatory cell chemotactic proteins in HSFs by IL-1α or TNFα suggests a cell-interactive mechanism for attracting inflammatory cells to the cornea in response to IL-1α or TNFα released from the epithelium by injury. One of these genes that appeared to be markedly upregulated was MCAF, also called MCP-1. This led us to study the expression of MCAF more completely both in vitro and in vivo. 

Upregulation of MCAF mRNA in HSFs in response to IL-1α or TNFα was confirmed using the quantitative RNase protection assay (Fig. 2A) . This experiment was repeated three times, and consistent results were obtained in each experiment. Thus, either IL-1α or TNFα upregulates the expression of the MCAF gene in HSFs in vitro. 

Western blot analysis was used to confirm MCAF protein production in HSFs (Fig. 2B) . Two isoforms of MCAF (approximately 19 and 16 kDa) were detected in HSFs, which compares favorably with the size of MCAF isoforms reported in a previous study. ¹⁵ Although quantitation with Western blot analysis is not completely reliable, there appeared to be an increase in both isoforms of MCAF in HSFs in response to 24 or 48 hours of IL-1α or TNFα exposure. This experiment was repeated three times, and the results were consistent between the experiments. 

The effect of scrape injury on MCAF protein expression in the rabbit cornea was examined with immunocytochemistry (Fig. 3) . No MCAF protein was detected in keratocytes in the unwounded cornea (Fig. 3A) . There was heavy staining at the apical surface of the epithelium, suggesting high levels of MCAF on the corneal surface. At 4 hours (Fig. 3B) and 24 hours (Fig. 3C) after corneal epithelial scrape injury there was marked upregulation of MCAF protein expression in the keratocytes in the posterior stroma. There was little MCAF detected in the anterior stroma, consistent with apoptosis of anterior keratocyte cells that occurs after corneal epithelial scrape injury. ⁶ This experiment was repeated three times with identical results each time. 

Large numbers of cells that were positive for the monocyte-macrophage–specific antigen recognized by the T2008X antibody were detected by immunocytochemistry in the corneal stroma at 24 hours after corneal epithelial scrape injury, but not in control unwounded corneas (Fig. 4) . No cells were detected with isotypic control antibody (Fig. 4) or when primary antibody was omitted (not shown). These cells were difficult or impossible to detect in the anterior stroma of the wounded cornea using hematoxylin and eosin staining (not shown), as has been previously noted. ^{6 14}  

Microinjection of 100 ng MCAF into the stroma of the mouse cornea resulted in the appearance of large numbers of uniform cells into the stroma at 24 or 48 hours after microinjection (Fig. 5A) . Microinjection of 100 ng of G-CSF into the stroma of the mouse cornea resulted in the appearance of large numbers of cells with the typical morphology of granulocytes at 24 or 48 hours after microinjection (Fig. 5B) . Microinjection of vehicle control solution had no effect on the stroma (Fig. 5C) . 

Discussion

Changes in the expression and localization of key cytokines and receptors are probably important in wound healing and homeostasis in the cornea. IL-1α appears to be especially important, because it regulates many processes that are integral to the response to injury. These include keratocyte apoptosis, ^{6 7} expression of hepatocyte growth factor and keratinocyte growth factor production by keratocytes and myofibroblasts, ^{5 8} and keratocyte collagenase and metalloproteinase production. ^{9 10 11} IL-1α is produced constitutively in the corneal epithelium and is released by injury or death of the epithelial cells. ^{1 2 3 4 6} IL-1α is not detectable in keratocytes in the unwounded cornea. ⁴ IL-1α released from injured corneal epithelial cells triggers an autocrine loop in viable keratocytes resulting in production of IL-1α in the stroma of the acutely wounded cornea. ¹⁰ The wide range of effects controlled by IL-1α have led us to consider it a master regulator of the response to acute corneal injury. TNFα is probably also released after injury to the epithelium and may contribute to the response to wounding. ¹²  

In this study, we examined the effects of IL-1α and TNFα on the expression of a number of cytokines, chemokines, and receptors in cultured human corneal stromal fibroblasts, by using gene array technology. Several cytokines, chemokines, and receptors were upregulated in stromal fibroblasts in response to IL-1α (Table 1) . A few were downregulated by IL-1α (Table 2) . Similarly, TNFα modulated the expression of several cytokine, chemokine, and receptor genes in stromal fibroblasts (Tables 3 4) . The effects of IL-1α and TNFα were similar for some of the cytokine and receptor genes represented on the array. IL-1α or TNFα, however, had specific effects on other cytokine and receptor genes evaluated by gene array technology. 

A striking aspect of the results from the array experiments was the number of genes upregulated in stromal fibroblasts by IL-1α and/or TNFα (of the 268 total represented on the array) that function in the chemotaxis, proliferation, differentiation, and activation of inflammatory cells. Thus, G-CSF, ^{16 17} MCAF, ^{18 19} neutrophil-activating peptide (ENA-78), ^{20 21} monocyte-derived neutrophil chemotactic factor (MDNCF or IL-8), ^{22 23} and interleukin-4 ^{24 25 26} were all upregulated in stromal fibroblasts by IL-1α. G-CSF and neutrophil-activating peptide (ENA-78) were upregulated by TNFα. These and other inflammatory cytokines and chemokines that were upregulated are indicated by bold lettering in Tables 1 and 3 . 

Upregulation of these cytokines in cultured stromal fibroblasts in response to IL-1α and TNFα is of particular interest, because they could function as the modulators that trigger the known inflammatory cell infiltration that occurs in vivo approximately 24 hours after corneal epithelial scrape injury. ²⁷ In the current study, we detected a large number of cells expressing monocyte-macrophage–specific antigen in the corneal stroma at 24 hours after corneal epithelial scrape injury, consistent with this hypothesis (Fig. 4) . In addition, another recent study used electron microscopy to show that large numbers of polymorphonuclear cells infiltrate the corneal stroma at 24 and 72 hours after epithelial injury (Mohan et al., unpublished data, 2001). 

MCAF ^{18 19} was one of the cytokines that was markedly upregulated in stromal fibroblasts by IL-1α or TNFα. A similar result was reported in a previous study for MCAF in vitro. ²⁸ That study and the current one suggest a cytokine-mediated mechanism leading to the chemotactic attraction of inflammatory cells, such as monocytes or macrophages, which infiltrate the stroma approximately 24 hours after corneal epithelial injury. Consistent with this hypothesis, keratocytes expressed MCAF protein detected by immunocytochemistry at 4 and 24 hours after corneal epithelial scrape injury (Fig. 3) . No MCAF was detected in the keratocytes in the unwounded cornea (Fig. 3) . Cells expressing monocyte-macrophage–specific antigen appeared in the cornea after epithelial scrape injury (Fig. 4) . Finally, microinjection of MCAF into the corneal stroma triggered the influx of inflammatory cells that had morphology consistent with monocytes-macrophages (Fig. 5) . 

Cultured stromal fibroblasts produced low levels of MCAF and other proinflammatory cytokines and chemokines in the absence of IL-1α or TNFα (Figs. 1 2A 2B) . In the case of MCAF, no production was noted in the unwounded cornea in vivo. Thus, MCAF production in keratocytes in vivo was detected only after the epithelium was injured. Some investigators have noted that cultured stromal fibroblasts are more similar to wound-healing fibroblasts than the keratocytes in the unwounded cornea in vivo. ^{29 30} Thus, the production of MCAF and the other cytokines and chemokines (Fig. 1) in cultured stromal fibroblasts in the absence of IL-1α or TNFα may be more representative of the keratocyte-derived cells in the wounded cornea (i.e., myofibroblasts or wound-healing fibroblasts) than of keratocytes in vivo. 

G-CSF was also upregulated in stromal fibroblasts by IL-1α or TNFα. Microinjection of G-CSF triggered the influx of cells into the stroma that had the morphology of granulocytes (Fig. 5) . Thus, G-CSF may have an important role in regulating the influx of granulocytes into the cornea. 

Cytokine-mediated induction of proinflammatory chemokine production by keratocytes may have an important role in modulating the influx of inflammatory cells into the cornea. Such communications are probably important in many corneal conditions associated with inflammatory cell infiltration, including infection, contact lens–induced sterile infiltrates, and the inflammatory cell influx associated with refractive surgical procedures, such as LASIK and photorefractive keratectomy. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2000.

 Supported in part by Grants EY10056 and EYO1730 from the National Eye Institute and an unrestricted grant from Research to Prevent Blindness.

 Submitted for publication February 13, 2001; revised May 9 and July 13, 2001; accepted July 24, 2001.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 Corresponding author: Steven E. Wilson, Department of Ophthalmology, University of Washington School of Medicine, Box 356485, Seattle, WA 98195-6485.

 

Figure 1.

 

Representative cytokine–chemokine gene arrays hybridized with labeled RNA probe isolated from cultured HSFs exposed to vehicle control (A), 20 ng/ml IL-1α (B), or 20 ng/ml TNFα (C) for 6 hours. This is a representative film exposure time of 48 hours. Arrows: MCAF gene in all three grids from the same experiment. Quantitation of this gene would be performed at a lower exposure, at which the signal is not as diffuse, but this 48-hour exposure time provided an overall perspective of the alterations in expression that were noted in this experiment. Note the difference in signal for the MCAF gene among control, IL-1α, and TNFα. Similar differences were noted for other genes. The quantitative results are provided in Tables 1 2 3 4 . The identity of individual genes corresponding to the spot pairs can be determined at http://www.clontech.com.

Table 1.

 

List of Upregulated Genes in IL-1α–Treated Human Stromal Fibroblasts

Matrix Location	Accession Number	Intensity Difference	Protein/Gene
6J	X03438	27920	Granulocyte colony-stimulating factor (G-CSF)
11O	M31159	26295	Growth hormone-dependent insulin-like growth factor-binding protein
18B	M96955	25904	Teratocarcinoma-derived growth factor 1 (TDGF1)
3F	M13982	24199	Interleukin-4 (IL-4)
7D	M24545	24005	Monocyte chemotactic and activating factor (MCAF), MCP-1
16F	J03634	23842	Erythroid differentiation protein (EDF)
22L	D10923	22936	HM74
4E	U32659	22879	Interleukin-17 (IL-17)
9I	L08187	22005	Cytokine receptor (EB13)
10K	U14187	21430	Receptor tyrosine kinase ligand LERK-3 (EPLG3)
19G	U02687	19812	Growth factor receptor tyrosine kinase (STK-1)
23L	M83941	19740	Tyrosine kinase receptor (HEK)
3I	J04156	18665	Interleukin-7 (IL-7)
4F	M63099	18157	Interleukin-1 receptor antagonist
6C	X72755	18115	Cytokine humig; interferon-γ–induced monokine
3H	X04602	17435	Interleukin-6 (IL-6) precursor; B-cell stimulatory factor 2 (BSF-2)
3K	X17543	15858	Interleukin-9 (IL-9); (P40)
6E	X14454	15639	Interferon regulatory factor 1
22H	U06863	15252	Follistatin-related protein precursor
7E	X78686	14881	Neutrophil-activating peptide (ENA-78)
23K	X74764	14804	Tyrosine kinase receptor (TKT)
5E	M84747	14519	Interleukin-9 receptor
5D	M68932	13903	Interleukin-8 receptor α (IL8RA)
8E	X17648	13382	Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFRa)
9D	X53655	13296	Nerve growth factor-2 (NT-3)
3E	M14743	12896	Interleukin-3 (IL-3) precursor
3J	Y00787	12587	Monocyte-derived neutrophil chemotactic factor (MDNCF or IL-8)

 Bold type indicates upregulated cytokines and chemokines.

Table 2.

 

List of Downregulated Genes in IL-1α–Treated Human Stromal Fibroblasts

Matrix Location	Accession Number	Intensity Difference	Protein/Gene
17F	X66945	−13540	Basic FGF receptor-1 precursor; (EC 2.7.1.112, fms-like tyrosine kinase-2, C-FGR, FGFR1)
4G	M27492	−13909	Interleukin-1 receptor
1I	U14971	−14119	40S ribosomal protein S9
15C	U41745	−14833	PDGF-associated protein
23G	M62424	−15807	Coagulating factor II receptor
1E	K00558	−17015	Brain-specific tubulin-α-1 subunit (TUBA1)
18F	X00588	−17311	Epidermal growth factor receptor
9K	M14764	−17888	Nerve growth factor receptor
1H	X56932	−17942	23-kDa Highly basic protein; 60S ribosomal protein L13A (RPL 13A)
1D	X01677	−17949	Liver glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
23H	M92381	−18532	Thymosin-β-10
1F	M11886	−18678	HLA class I histocompatibility antigen C-4 α-subunit (HLAC)
1A	M26880	−18779	Ubiquitin
9E	X52946	−18798	Nerve growth factor HBNF-1; pleiotrophin precursor (PTN);
9G	M76125	−18928	AXL tyrosine kinase receptor
1C	V00530	−19374	Hypoxanthine-guanine phosphoribosyltransferase (HPRT)
15G	M19154	−20554	Transforming growth factor-β-2; glioblastoma-derived T-cell suppressor factor
5B	M57230	−22488	Glycoprotein gp130
12B	M62403	−23323	IGFBP4
15D	M21574	−26215	PDGF-α receptor

Table 3.

 

List of Upregulated Genes in TNF-α–Treated Human Stromal Fibroblasts

Matrix Location	Accession Number	Intensity Difference	Protein/Gene
6J	X03438	27588	Granulocyte colony-stimulating factor (G-CSF)
11O	M31159	24514	Growth hormone-dependent insulin-like growth factor-binding protein
16F	J03634	22612	Erythroid differentiation protein (EDF)
18B	M96955	20699	Teratocarcinoma-derived growth factor 1 (TDGF1)
10K	U14187	20652	Receptor tyrosine kinase ligand LERK-3 (EPLG3)
9I	L08187	20342	Cytokine receptor (EB13)
22L	D10923	20155	HM74
4E	U32659	18683	Interleukin-17 (IL-17)
6C	X72755	17216	Cytokine humig; interferon-γ–induced monokine
5E	M84747	17015	Interleukin-9 receptor
19G	U02687	14871	Growth factor receptor tyrosine kinase (STK-1)
14L	M31165	13856	TNF-inducible hyaluronate-binding protein (TSG-6)
23K	X74764	13520	Tyrosine kinase receptor (TKT)
7E	X78686	13486	Neutrophil-activating peptide (ENA-78)
22H	U06863	12969	Follistatin-related protein precursor

 Bold type indicates upregulated cytokines and chemokines.

Table 4.

 

List of Downregulated Genes in TNF-α–Treated Human Stromal Fibroblasts

Matrix Location	Accession Number	Intensity Difference	Protein/Gene
15C	U41745	−14275	PDGF associated protein
9G	M76125	−14618	AXL tyrosine kinase receptor
1I	U14971	−16048	40S ribosomal protein S9
20H	L20861	−16303	Wnt-5a
9K	M14764	−17554	Nerve growth factor receptor
1E	K00558	−17837	Brain-specific tubulin α 1 subunit (TUBA1)
5B	M57230	−18306	Glycoprotein gp130
22B	M77349	−18915	BIGH3
1G	X00351	−18987	Cytoplasmic beta-actin (ACTB)
1D	X01677	−19060	Liver glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
1H	X56932	−19180	23-kDa Highly basic protein; 60S ribosomal protein L13A (RPL 13A)
9E	X52946	−19530	Nerve growth factor HBNF-1; pleiotrophin precursor (PTN)
4G	M27492	−20022	Interleukin-1 receptor
1A	M26880	−20655	Ubiquitin
23H	M92381	−20741	Thymosin-β-10
12B	M62403	−21434	IGFBP4
1F	M11886	−21639	HLA class I histocompatibility antigen C-4 α-subunit (HLAC)
1C	V00530	−23408	Hypoxanthine-guanine phosphoribosyltransferase (HPRT)
15G	M19154	−26753	Transforming growth factor β-2; glioblastoma-derived T-cell suppressor factor

Figure 2.

 

MCAF mRNA and protein production in cultured HSFs stimulated with IL-1α, TNFα, and Fas-activating IgM (Fas Ab). (A) Quantitative RNase protection assay revealed marked upregulation of MCAF mRNA in response to TNFα or IL-1α stimulation for 6 hours compared with the control. To a lesser extent, the Fas-activating antibody also stimulated MCAF production in cells that remained viable in response to this apoptosis-stimulating receptor activation. The size of the protected band was 253 bp. This experiment was repeated three times with consistent results. (B) MCAF protein was detected by Western blot analysis in the conditioned culture medium of HSFs treated for 24 or 48 hours with IL-1α, TNFα, or the Fas-activating antibody, compared with control cultures. Markers are shown in kilodaltons in the lane on the left. The two isoforms of MCAF were detected in each culture. They appeared to be present at higher levels in cells treated with IL-1α or TNFα, because identical amounts of protein were applied in each lane. A unique 35-kDa protein that bound the MCAF antibody was identified only in cultures treated with the Fas-activating antibody. This protein has not been characterized.

Figure 3.

 

Detection of MCAF protein in rabbit cornea using immunocytochemistry. (A) No MCAF protein was detected in the keratocytes in the unwounded cornea. MCAF or MCAF-like protein was detected at the corneal epithelial surface (arrow) in the unwounded cornea. This protein may have been derived from the tears. At 4 hours (C) and 24 hours (E) after epithelial scrape injury there was upregulation of MCAF protein in keratocytes in the posterior stroma of the central cornea (arrows). Expression in anterior keratocytes was not detected because most underwent apoptosis by 4 hours after epithelial scrape injury. ⁴ No MCAF protein was detected with an isotypic control primary antibody at the same time points (B, D, and F, respectively). Wounded corneas had stromal swelling because there was no epithelial barrier function. Magnification, ×400.

Figure 4.

 

Detection of monocytes-macrophages in the stroma of the rabbit cornea at 24 hours after corneal epithelial scrape injury by immunocytochemistry with an antibody specific for these cells. In 24-hour wounded corneas shown in (A) and at higher magnification in (B), cells that stained with the monocyte-macrophage–specific antigen recognized by the T2008X antibody (arrows) were detected in the stroma at 24 hours after epithelial scrape injury. No cells were detected with an isotypic control antibody or without primary antibody (not shown). In unwounded corneas shown in (C) and at higher magnification in (D), no cells were detected with this antibody in the stroma. Magnification, (A, C) ×200; (B, D) ×400.

Figure 5.

 

Microinjection of chemokines into the mouse cornea triggered inflammatory cell infiltration into the stroma. (A) Microinjection of 100 ng mouse MCAF triggered the influx of large numbers of round mononuclear cells (arrowheads). (B) Microinjection of 100 ng mouse G-CSF triggered the influx of large numbers of inflammatory cells. Many of these cells had multilobular nuclei (arrowheads) characteristic of granulocytes. (C) Microinjection of vehicle control had no effect on the corneal stroma. Magnification, ×400.

The authors thank Jing Huang and Dan Possin for assistance with the immunocytochemistry experiments.