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Figure 1.
Leukocyte subsets in inflamed corneas determined by immunohistologic staining with a 3-step streptavidin-peroxidase method. Values shown are mean ± SEM of 10 corneal lesions. PMN indicates polymorphonuclear leukocytes.

Leukocyte subsets in inflamed corneas determined by immunohistologic staining with a 3-step streptavidin-peroxidase method. Values shown are mean ± SEM of 10 corneal lesions. PMN indicates polymorphonuclear leukocytes.

Figure 2.
Chemokine messenger RNA (m RNA)expression in inflamed peripheral cornea (limbus). A and B, Immunostaining using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate, showing infiltration of CD68+ macrophages from peripheral cornea (A) and neutrophils (PMN) in central cornea (B). C-G, In situ hybridization for m RNA expression with sulfur 35–uridine triphosphate–labeled RNA antisense probes, showing high growth-related oncogene-α (Gro-α)m RNA expression in limbal epithelium (C) and macrophage chemoattractant protein 1 (MCP-1) m RNA expression in basal limbal epithelium and subepithelial cells(D [dark-field illumination] and E); MCP-1 expression stops from transition of limbal to corneal epithelium (arrow) (E); focal expression of CC chemokine regulated on activation, normal T-cell expressed and secreted (RANTES) in limbal epithelium (F); and expression of CXC chemokine macrophage interferon-γ inducible gene (MIG; arrows) in single epithelial cells (G) (original magnification, ×100 [A-E], ×400 [F, G]).

Chemokine messenger RNA (m RNA)expression in inflamed peripheral cornea (limbus). A and B, Immunostaining using a 3-step streptavidin-peroxidase method and 3-amino-9-ethyl-carbazole as substrate, showing infiltration of CD68+ macrophages from peripheral cornea (A) and neutrophils (PMN) in central cornea (B). C-G, In situ hybridization for m RNA expression with sulfur 35–uridine triphosphate–labeled RNA antisense probes, showing high growth-related oncogene-α (Gro-α)m RNA expression in limbal epithelium (C) and macrophage chemoattractant protein 1 (MCP-1) m RNA expression in basal limbal epithelium and subepithelial cells(D [dark-field illumination] and E); MCP-1 expression stops from transition of limbal to corneal epithelium (arrow) (E); focal expression of CC chemokine regulated on activation, normal T-cell expressed and secreted (RANTES) in limbal epithelium (F); and expression of CXC chemokine macrophage interferon-γ inducible gene (MIG; arrows) in single epithelial cells (G) (original magnification, ×100 [A-E], ×400 [F, G]).

Figure 3.
Expression of neutrophil and mononuclear cell attractant CC and CXC chemokines in corneal lesions. Values shown are mean ± SEM of 10 corneal lesions. In situ hybridization was carried out with chemokine-specific sulfur 35–UTP–labeled RNA antisense probes as described in the "Methods" section. MCP-1 indicates macrophage chemoattractant protein 1; MIP-1α, macrophage inflammatory protein 1α; RANTES, regulated on activation, normal T-cell expressed and secreted; Gro-α, growth-related oncogene-α; IL-8, interleukin 8; IP-10, interferon-γ inducible protein 10; and MIG, macrophage interferon-γ inducible gene.

Expression of neutrophil and mononuclear cell attractant CC and CXC chemokines in corneal lesions. Values shown are mean ± SEM of 10 corneal lesions. In situ hybridization was carried out with chemokine-specific sulfur 35–UTP–labeled RNA antisense probes as described in the "Methods" section. MCP-1 indicates macrophage chemoattractant protein 1; MIP-1α, macrophage inflammatory protein 1α; RANTES, regulated on activation, normal T-cell expressed and secreted; Gro-α, growth-related oncogene-α; IL-8, interleukin 8; IP-10, interferon-γ inducible protein 10; and MIG, macrophage interferon-γ inducible gene.

Figure 4.
Chemokine messenger RNA (m RNA)expression by in situ hybridization with sulfur 35–uridine triphosphate–labeled RNA antisense probes in central inflamed cornea. There is high growth-related oncogene-α (Gro-α) m RNA expression in central corneal epithelium(A) and in corneal stroma (D). Interleukin 8 (IL-8) m RNA expression is absent in corneal epithelium but present in keratocytes in central stroma (B). There is a high number of macrophage chemoattractant protein 1 (MCP-1) m RNA-positive stromal cells in corneal stroma (C) and low macrophage interferon-γ inducible gene (MIG; arrows) (E) and macrophage inflammatory protein 1α(MIP-1α) (F) m RNA expression in corneal keratocytes (original magnification, ×400 [A-C, E, F], ×320 [D]).

Chemokine messenger RNA (m RNA)expression by in situ hybridization with sulfur 35–uridine triphosphate–labeled RNA antisense probes in central inflamed cornea. There is high growth-related oncogene-α (Gro-α) m RNA expression in central corneal epithelium(A) and in corneal stroma (D). Interleukin 8 (IL-8) m RNA expression is absent in corneal epithelium but present in keratocytes in central stroma (B). There is a high number of macrophage chemoattractant protein 1 (MCP-1) m RNA-positive stromal cells in corneal stroma (C) and low macrophage interferon-γ inducible gene (MIG; arrows) (E) and macrophage inflammatory protein 1α(MIP-1α) (F) m RNA expression in corneal keratocytes (original magnification, ×400 [A-C, E, F], ×320 [D]).

1.
Chandler  JWGillette  TE Immunologic defense mechanisms of the ocular surface. Ophthalmology. 1983;90585- 591Article
2.
Nelson  PJKrensky  AM Chemokines, chemokine receptors, and allograft rejection. Immunity. 2001;14377- 386Article
3.
Rossi  DZlotnik  A The biology of chemokines and their receptors. Annu Rev Immunol. 2000;18217- 242Article
4.
Luster  AD Chemokines--chemotactic cytokines that mediate inflammation. N Engl J Med. 1998;12436- 445
5.
Larson  CGAndersen  AOApella  EOppenheim  JJMatsushima  K The neutrophil activating protein (NAP-1) is also chemotactic for T-lymphocytes. Science. 1989;2431464- 1466Article
6.
Jinquan  TFrydengerg  JMukaida  N  et al.  Recombinant human growth-regulated oncogene-α induces T lymphocyte chemotaxis. J Immunol. 1995;1555359- 5368
7.
Loetscher  MGerber  BLoetscher  P  et al.  Chemokine receptor specific for IP-10 and MIG: structure, function and expression in activated T-lymphocytes. J Exp Med. 1996;184963- 969Article
8.
Schall  TJBacon  KToy  KJGoeddel  DV Selective attraction of monocytes and T-lymphocytes of the memory phenotype by cytokine RANTES. Nature. 1990;347669- 671Article
9.
Taub  DDConlon  KLloyd  AROppenheim  JJKelvin  DJ Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1α and MIP-1β. Science. 1993;260355- 358Article
10.
Carr  MWRoth  SJLuther  ERose  SSSpringer  TA MCP-1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A. 1994;913652- 3656Article
11.
Uguccioni  MD'Apuzzo  MLoetscher  MDewald  BBaggiolini  M Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 α and MIP-1 β on human monocytes. Eur J Immunol. 1995;2564- 68Article
12.
Elner  VMStrieter  RMPavilack  MA  et al.  Human corneal interleukin-8, IL-1 and TNF-induced gene expression and secretion. Am J Pathol. 1991;139977- 988
13.
Cubitt  CLTang  QMonteiro  CALausch  RNOakes  JE IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes. Invest Ophthalmol Vis Sci. 1993;343199- 3206
14.
Cubitt  CLLausch  RNOakes  JE Differential induction of GRO-α gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines. Invest Ophthalmol Vis Sci. 1997;381149- 1158
15.
Tran  MTTellaetxe-Isusi  MElner  VStrieter  RMLausch  RNOakes  JE Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. Invest Ophthalmol Vis Sci. 1996;37987- 996
16.
Farber  JM Hu MIG: a new member of the chemokine family of cytokines. Biochem Biophys Res Commun. 1993;192223- 230Article
17.
Liao  FRabin  RLYanelli  JRKoniaris  LGVanguri  PFarber  JM Human Mig chemokine: biochemical and functional characterization. J Exp Med. 1995;1821301- 1314Article
18.
Luster  ADRavetch  JV Biochemical characterization of a γ-interferon inducible cytokine(IP-10). J Exp Med. 1987;1661084- 1097Article
19.
Vanguri  PFarber  JM Identification of CRG-2: an interferon inducible m RNA predicted to encode a murine monokine. J Biol Chem. 1990;26515049- 15057
20.
Anisowicz  ABardwell  LSager  R Constitutive overexpression of a GROwth-regulated gene in transformed Chinese hamster and human cells. Proc Natl Acad Sci U S A. 1987;847188- 7192Article
21.
Richmond  ABalentien  EThomas  HG  et al.  Molecular characterization and chromosomal mapping of melanoma GROwth stimulatory activity, a GROwth factor structurally related to β-thromboglobulin. EMBO J. 1988;72025- 2033
22.
Yoshimura  TMatsushima  KTanaka  S  et al.  Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc Natl Acad Sci U S A. 1987;849233- 9237Article
23.
Yoshimura  TYuhki  NMoore  SKAppella  ELerman  MILeonard  EJ Human monocyte chemoattractant protein-1 (MCP-1): full-length c DNAcloning, expression in mitogen-stimulated blood mononuclear leukocytes and sequence similarity to mouse competence gene JE. FEBS Lett. 1989;244487- 493Article
24.
Schall  TJJongstra  JDyer  B  et al.  Human T-cell specific molecule is a member of a new gene family. J Immunol. 1988;1411018- 1025
25.
Wolpe  SDDavatelis  GSherry  B  et al.  Macrophages secrete a novel heparin binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med. 1988;167570- 581Article
26.
Hong  JWLiu  JJLee  JS  et al.  Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci. 2001;422795- 2803
27.
Gillitzer  RWolff  KTong  D  et al.  MCP-1 m RNA expression in basal keratinocytes of psoriatic lesions. J Invest Dermatol. 1993;101127- 131Article
28.
Gillitzer  RRitter  USpandau  UGoebeler  MBröcker  EB Differential expression of GRO-α and IL-8 m RNA in psoriasis: a model for neutrophil migration and accumulation in vivo. J Invest Dermatol. 1996;107778- 782Article
29.
Goebeler  MToksoy  ASpandau  UEngelhardt  EBröcker  EBGillitzer  R The CXC chemokine MIG is highly expressed in the papillae of psoriatic lesions. J Pathol. 1998;18489- 95Article
30.
Spandau  UToksoy  AGoebeler  MGillitzer  R MIG is a dominant lymphocyte-attractant chemokine in lichen planus lesions. J Invest Dermatol. 1998;1111003- 1009Article
31.
Spandau  UBröcker  EBKämpgen  EGillitzer  R C-C and C-X-C chemokines are differentially expressed in erythema multiforme in vivo. Arch Dermatol. 2002;1381027- 1033Article
32.
Engelhardt  EToksoy  AGoebeler  MDebus  SBroecker  EBGillitzer  R Chemokines IL-8, Gro-α, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am J Pathol. 1998;1531849- 1860Article
33.
Wilson  SEHe  YGWeng  J  et al.  Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res. 1996;62325- 327Article
34.
Planck  SRRich  LFAnsel  JCHuang  XNRosenbaum  JT Trauma and alkali burns induce distinct patterns of cytokine gene expression in the rat cornea. Ocul Immunol Inflamm. 1997;595- 100Article
35.
Kennedy  MKim  KHHarten  B  et al.  Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci. 1997;382483- 2491
36.
Taylor  PC Anti-TNF therapy for rheumatoid arthritis and other inflammatory diseases. Mol Biotechnol. 2001;19153- 168Article
37.
Sfikakis  PP Behcet's disease: a new target for anti-tumour necrosis factor treatment. Ann Rheum Dis. 2002;61 ((suppl 2)) II51- II53
38.
Arend  WP The mode of action of cytokine inhibitors. J Rheumatol Suppl. 2002;6516- 21
39.
Tsuji  FSawa  KKato  MMibu  HShirasawa  E The effects of betamethasone derivatives on endotoxin-induced uveitis in rats. Exp Eye Res. 1997;6431- 36Article
Laboratory Sciences
June 2003

High Expression of Chemokines Gro-α (CXCL-1), IL-8 (CXCL-8), and MCP-1 (CCL-2) in Inflamed Human Corneas In Vivo

Author Affiliations

From the Department of Ophthalmology, Clinical Faculty of the University of Heidelberg, Mannheim, Germany (Dr Spandau); Department of Dermatology, University of Wuerzburg, Wuerzburg, Germany (Ms Toksoy and Dr Gillitzer); Department of Pathology, University of Witwatersrand, Johannesburg, South Africa (Dr Verhaart); and Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany (Dr Kruse). The authors have no relevant financial interest in this article.

Arch Ophthalmol. 2003;121(6):825-831. doi:10.1001/archopht.121.6.825
Abstract

Objective  To investigate in vivo expression of chemokines in normal and inflamed human corneas, to determine whether chemokines are responsible for the recruitment of inflammatory cells.

Methods  In situ hybridization of the CXC chemokines growth-related oncogene-α(Gro-α) (CXCL-1), interleukin 8 (CXCL-8), macrophage interferon-γ inducible gene (CXCL-9), and interferon-γ inducible protein 10 (CXCL-10) and of the CC chemokines macrophage chemoattractant protein 1 (MCP-1) (CCL-2), macrophage inflammatory protein 1α (CCL-3), and regulated on activation, normal T-cell expressed and secreted (CCL-5) was performed to localize chemokine messenger RNA. Immunohistochemistry was used to identify the cellular infiltrate within the cornea. Three normal human eyes were compared with eyes enucleated because of chronic inflammation (n = 10), secondary to perforating injuries.

Results  In normal corneas, no chemokine expression was detected. In inflamed lesions, a high intensity of signals from Gro-α (CXCL-1) and MCP-1 (CCL-2) messenger RNA was observed in limbal epithelium and from Gro-α (CXCL-1), interleukin 8 (CXCL-8), and MCP-1 (CCL-2) in corneal stroma. The Gro-α(CXCL-1) was the only chemokine expressed by central corneal epithelium. All other examined chemokines were only moderately expressed in limbus and corneal stroma, or barely detectable.

Conclusions  These cytokines are important agents in the cytokine network and contribute to the cell-specific and spatially restricted recruitment of neutrophils and mononuclear cells in acute inflammatory lesions of the human cornea.

Clinical Relevance  Understanding the role of chemokines in corneal inflammation may lead to the development of a selective receptor blockage of highly expressed chemokines to inhibit the recruitment of leukocyte subsets.

ONE OF THE FUNCTIONS of the ocular surface is to protect the eye from infectious agents. The ocular surface comprises several tissues: the cornea, the conjunctiva, and the transition zone between the two, the limbus. The cornea consists of 3 distinct layers (a stratified epithelium, a dense stroma with a network of keratocytes, and a single-layered endothelium) and is characterized by an almost complete absence of white blood cells during physiologic conditions. In contrast, the limbus is known for its immunologic functions and harbors a variety of inflammatory cells.1 Although the infiltration of leukocytic cells is a common defense mechanism of the ocular surface, 1 we do not know what factors are responsible for the presence of leukocytes in inflamed corneal tissue. Among the possible mediators for leukocyte recruitment are a subgroup of cytokines with chemoattractive properties for leukocyte subsets, called chemokines (chemo tactic cytokines).24 Chemokines are thought to play a crucial role in inflammatory diseases and are subdivided into 2 groups.3 Chemokines of the CXC subgroup(α-chemokines) are, according to in vitro studies, potent chemoattractants for neutrophils and T lymphocytes, 57 and chemokines of the CC subgroup (β-chemokines) are known for their chemotactic properties toward macrophages and T-cell subsets.811

Several studies have demonstrated the expression of CC and CXC chemokines in human corneal epithelial cells and keratocytes in vitro.1215 However, few data exist examining the expression of chemokines in human cornea in vivo.12 We, therefore, examined the expression of 4 members of the α-chemokine family—macrophage interferon-γ inducible gene (MIG, CXCL-9), 16,17 interferon-γ inducible protein 10 (IP-10, CXCL-10), 18,19 growth-related oncogene-α (Gro-α, CXCL-1), 20,21 and interleukin 8 (IL-8, CXCL-8)22—and of 3 members of the β-chemokine family—macrophage chemoattractant protein 1 (MCP-1, CCL-2), 23 regulated on activation, normal T-cell expressed and secreted (RANTES, CCL-5), 24 and macrophage inflammatory protein 1α (MIP-1α, CCL-3)25—in inflamed corneal lesions in vivo. These chemokines are well known for their chemotactic properties toward neutrophils, macrophages, and lymphocytes.24 We studied their chemokine profile in inflamed human corneas secondary to penetrating injuries of the eyeball (n = 10) with an infiltrate consisting of neutrophils and mononuclear leukocytes.

METHODS
SELECTION AND PROCESSING OF HUMAN EYES

Normal human corneas (n = 3) were obtained from eyes without anterior segment abnormalities that were enucleated because of malignant melanoma of the choroid. The inflamed corneas (n = 10) were obtained from eyes that were enucleated an average of 2 to 4 days after penetrating injuries of the scleral wall to prevent sympathetic uveitis. The causes of rupture of the eyeball were sharp penetrating trauma caused by glass (n = 2), sharp or blunt metal objects (n = 5), and unknown objects (n = 3). Specimens were embedded in formalin and paraffinized. Four-micrometer serial sections were fixed at high temperature for immunohistochemistry (30 minutes, 52°C) and for in situ hybridization(12 hours, 52°C).

IMMUNOHISTOLOGY

For immunohistologic analyses, the following mouse monoclonal antibodies were used: anti-CD1a recognizing Langerhans cells (Beckman Coulter, Krefeld, Germany); anti-CD3 reacting with the T-cell receptor–associated CD3 antigen (Becton, Dickinson and Company, Sunnyvale, Calif); anti-CD68 recognizing monocytes and macrophages (DAKO Corp, Hamburg, Germany); and anti–neutrophil elastase detecting neutrophils (DAKO Corp). Biotin-conjugated sheep anti–mouse immunoglobulin (Amersham, Braunschweig, Germany) and biotin-conjugated mouse anti–goat immunoglobulin (Jackson Immuno Research Laboratories, Inc, West Grove, Pa) were used as second-stage reagents. For immunohistologic staining, a 3-step streptavidin–biotin peroxidase procedure was used as described.26 Briefly, after Fc receptors were blocked with 20% sheep serum in phosphate-buffered saline containing 5% skim milk powder and 0.1% Tween 20 (p H adjusted to 7.4), sections were incubated with the first-step antibody overnight at 4°C, followed by incubation with the second-step antibody for 1 hour at room temperature and preformed streptavidin–biotin peroxidase complex (Strept ABC-Peroxidase Complex; DAKO Corp) (1 hour, room temperature). Finally, sections were visualized by means of 3-amino-9-ethylcarbazole(AEC; Sigma-Aldrich, Taufkirchen, Germany) as peroxidase substrate. For control purposes, the first-step antibody was omitted and replaced by an irrelevant isotype-matched immunoglobulin or control serum. The controls consistently yielded negative results.

IN SITU HYBRIDIZATION

Preparation of sulfur 35 (35S)–labeled RNA probes was performed as described.26 The complementary DNA probes used for in situ hybridization were kindly provided by Tiezo Yoshimura, PhD (National Cancer Institute, Frederick, Md; MCP-1), Thomas Schall, PhD(Divisions of Discovery Biology and Molecular Pharmacology, Chemo Centryx, Inc, San Carlos, Calif; MIP-1β, RANTES), Genetics Institute (Cambridge, Mass; MIP-1α), Joshua M. Farber, PhD (National Institutes of Health, Bethesda, Md; MIG), Reinhard Kulke, PhD (University of Kiel, Kiel, Germany; IP-10), Charles Weissmann, PhD (University of Zürich, Zürich, Switzerland; IL-8), and Anthony Anisowicz, PhD (Dana Farber Cancer Institute, Boston, Mass; Gro-α). Sequence analysis of the Gro-α probe used and comparison with Gro-β and Gro-γ proved the probe to be subtype specific (data not shown).2,20 After linearization of plasmid DNA with appropriate restriction enzymes, 35S-labeled sense and antisense probes were obtained by in vitro transcription with the use of SP6, T3, or T7 RNA polymerases (Boehringer Mannheim, Mannheim, Germany) together with adenosine triphosphate, guanosine triphosphate, cytidine triphosphate(Boehringer Mannheim), and [35S]uridine triphosphate (Amersham) as substrates. After elimination of the original linearized template complementary DNA, alkaline hydrolysis of labeled probes was performed for 30 to 50 minutes. After several ethanol precipitation steps, the radioactive riboprobes were adjusted to a specific activity of 2 × 106 cpm/µL in 0.01M Tris hydrochloride, p H 7.5, containing 1m M EDTA.

Hybridization procedure was performed as described previously.26 Paraformaldehyde-fixed cryostat sections were treated with proteinase K (1 µg/mL; Boehringer Mannheim) for 30 minutes at 37°C, refixed in paraformaldehyde, acetylated with acetic anhydride in 0.1M triethanolamine(p H 98.0, 10 minutes), dehydrated, and air-dried. The sections were overlaid with 20 µL of hybridization solution (50% formamide, 300m M sodium chloride, 20m M Tris hydrochloride, p H 8.0, 5m M EDTA, 1× Denhardt solution, 10% dextran sulfate, 100m M dithiothreitol, and 2 × 105 cpm/µL heat-denaturated radioactive probe). After hybridization for 12 to 16 hours, nonhybridized probes were removed by several high-stringency washing procedures. Nonspecific background RNA was digested with RNase A1 (20 µL/mL) and RNase T1 (1 U/mL; Boehringer Mannheim) for 30 minutes at 37°C. For autoradiography, slides were dipped in a photo-emulsion (Kodak NTB-2, 1:2 in 800m M ammonium acetate; Eastman Kodak Company, Rochester, NY) and exposed for 4 weeks at 4°C.

EVALUATION OF SLIDES

For evaluation of the developed slides, a microscope (Axiophot; Carl Zeiss, Oberkochen, Germany) was used. Positive cells were counted with an ocular square grid at randomly selected areas (n = 5-8) of all (n = 10) corneal lesions (magnification ×200 and ×400) and related to the total number of cells. The average percentage of messenger RNA (m RNA)–expressing cells was determined as mean ± SEM.

This study was performed according to the guidelines of the Declaration of Helsinki.

RESULTS
HISTOLOGIC CHARACTERIZATION OF LEUKOCYTE SUBSETS IN CORNEAL LESIONS

For identification and localization of infiltrating leukocyte subsets, immunohistochemistry was performed on inflamed (n = 10) and normal (n = 3) corneas by means of a panel of monoclonal antibodies that recognize T lymphocytes, macrophages, neutrophils, and Langerhans cells. The inflamed lesions showed a mild to moderate inflammation corresponding to enucleation 2 to 4 days after trauma. None of the investigated specimens had stromal vascularization. All lesions had a more or less pronounced infiltrate localized below the limbal epithelium, and 3 lesions additionally demonstrated a leukocytic infiltration of central corneal stroma. Only single white blood cells were detected in corneal epithelium. The relative percentages of all leukocyte subtypes investigated depending on the location are summarized in Figure 1. The subepithelial limbal infiltrate was found to consist predominantly of polymorphonuclear leukocytes and, to a lesser extent, of mononuclear cells (Figure 2A) (more polymorphonuclear leukocytes than CD68+ macrophages, and more CD68+ macrophages than CD3+ T lymphocytes). The histologic characterization of the 3 lesions with infiltrated central stroma showed 1 lesion with CD68+ macrophages scattered in central cornea and 2 lesions with a polymorphonuclear leukocyte infiltration (Figure 2B). In all investigated specimens, CD1+ Langerhans cells were barely detectable.

EXPRESSION OF GRO-α, IL-8, AND MCP-1 IN INFLAMED CORNEA

On the basis of the immunohistologic findings, the chemokines suspected of exerting a chemotactic effect on T lymphocytes, macrophages, and neutrophils were examined. With the use of in situ hybridization, cell-associated silver grain precipitates were detected with radioactively labeled antisense probes, whereas, for control purposes, sense probes yielded consistently negative results. Furthermore, with the rare exception of single positive cells localized in the limbal epithelium, chemokine m RNA expression in normal corneas was not detectable.

In contrast, inflamed lesions showed expression of various chemokines. However, the expression pattern was not uniform but showed pronounced differences regarding the intensity of expression as well as the location. Figure 3 shows a detailed quantitation of the percentage of chemokinem RNA-expressing cells according to the location. The intensity and location differed depending on the chemokine subgroup. The MCP-1 (CCL-2) showed the highest m RNA expression level of the CC chemokines, whereas Gro-α (CXCL-1) and IL-8 (CXCL-8) showed the highest expression levels of the CXC chemokines. All other chemokines investigated (MIP-1α [CCL-3], RANTES [CCL-5], MIG[CXCL-9], and IP-10 [CXCL-10]) were either moderately expressed or barely detectable.

CHEMOKINE EXPRESSION IN THE LIMBUS

With respect to the location, the regional distribution of inflammatory cells on the ocular surface as well as the presence of microvasculature corresponded to pronounced differences in the chemokine expression pattern between the limbus and the central stroma. In all lesions, chemokine expression was higher in the limbus (limbal epithelium more than limbal stroma) than central cornea(Figure 3). Furthermore, chemokine expression in limbal epithelium was paralleled in all lesions with a more or less pronounced subepithelial infiltrate. Of all examined chemokines, only Gro-α (CXCL-1) and MCP-1 (CCL-2) showed high expression levels in limbal epithelium (Figure 3). More than 27% ± 4.5% (mean ± SEM) of total cells in limbal epithelium expressed Gro-α (CXCL-1) and 15% ± 2.6% expressed MCP-1 (CCL-2). The expression patterns of Gro-α (CXCL-1) and MCP-1 (CCL-2), however, differed: Gro-α (CXCL-1) m RNA–positive cells were restricted to suprabasal epithelial cell layers of the limbus (Figure 2C), whereas MCP-1 (CCL-2) m RNA expression was localized along the basal epithelium of the limbus and in the subepithelial infiltrate(Figure 2D and E). The MCP-1 (CCL-2)m RNA expression stopped at the transition from limbal to corneal epithelium(Figure 2E, see arrow). All other examined chemokines showed low expression levels in limbal epithelium, as shown in Figure 3. The RANTES (CCL-5) was expressed by basal limbal epithelium and adjacent leukocytes (Figure 2F). In contrast, MIG (CXCL-9) was expressed only by single keratinocytes in the basal limbal epithelium (Figure 2G).

EXPRESSION OF ONLY GRO-α IN CENTRAL CORNEAL EPITHELIUM

The most conspicuous result was the strong expression of Gro-α(CXCL-1) in inflamed limbus. Sequence analysis of the Gro-α probe proved to be subtype specific. Expression of Gro-α (CXCL-1) was higher in limbal epithelium than in central cornea (limbal epithelium more than corneal epithelium, which was more than corneal stroma) (Figure 3). Furthermore, Gro-α (CXCL-1) was the only chemokine of all investigated chemokines expressed by corneal epithelium (Figure 4A). As mentioned in the previous section, MCP-1 (CCL-2) was not expressed by central corneal epithelium (Figure 2E). Furthermore, IL-8 (CXCL-8) message was not detected in corneal epithelium but in keratocytes in corneal stroma (Figure 4B). Finally, high amounts of Gro-α (CXCL-1) and MCP-1(CCL-2) m RNA transcripts were localized in corneal stroma (Figure 4C and D). In contrast, only a few keratocytes expressed CXC chemokine MIG (CXCL-9) (Figure 4E) and CC chemokine MIP-1α (CCL-3) m RNA (Figure 4F). According to the different m RNA expression levels and patterns, cross-hybridization between highly homologous chemokine probes could be excluded.

NEUTROPHIL ATTRACTANT CHEMOKINE m RNA EXPRESSION IN ACUTE INFLAMED CORNEA

If the composition of inflammatory cells is influenced by selective recruitment and trafficking through chemokines, expression of chemokines with neutrophil attractant properties should dominate acute inflamed corneas. To confirm this hypothesis, the number of cells expressing chemokines with neutrophil attractant properties (Gro-α [CXCL-1], IL-8 [CXCL-8]) were compared with cells that express mononuclear cell properties (MIG [CXCL-9], IP-10 [CXCL-10], MCP-1 [CCL-2], MIP-1α [CCL-3], and RANTES [CCL-5]). As shown in Figure 2, many more cells expressed neutrophil attractant chemokines than mononuclear cell chemokines. It is well known that MIG (CXCL-9) and IP-10 (CXCL-10) selectively recruit T lymphocytes, correlating with the low presence of CD3+ cells in inflamed lesions. Thus, the differential chemokine m RNA expression with dominance of neutrophil attractant chemokines (Gro-α [CXCL-1] and IL-8 [CXCL-8] over MIG [CXCL-9], IP-10[CXCL-10], MCP-1 [CCL-2], MIP-1α [CCL-3], and RANTES [CCL-5]) clearly reflects the composition of the infiltrates showing a preponderance of neutrophil elastase–positive neutrophils.

COMMENT

The infiltration of leukocytes, particularly neutrophils, is an event common to various types of inflammation. Chemokines are known to mediate the migration of specific leukocyte subsets to an area of inflammation. In this study we attempted to elucidate which chemokines are responsible for the recruitment of neutrophils and mononuclear cells in inflamed corneas. Recent studies showed the expression of CC and CXC chemokines by keratocytes and corneal epithelial cells.1315 These experiments were performed in cell culture. Experimental data about the expression of chemokines, especially in human corneas in vivo, is limited.12 It remains largely unknown which layers of inflamed corneas produce chemokines in vivo and what their expression patterns are.12,26 Furthermore, chemokine studies on inflammatory diseases show that cooperative efforts of a network of chemokines are important for leukocyte recruitment.24 However, a comprehensive repertoire of CXC and CC chemokines has not been investigated in an acute inflamed human cornea with a predominant neutrophil infiltrate. We therefore studied the chemokine profile in inflamed human corneas (n = 10) with an infiltrate consisting of polymorphonuclear cells.

The current study demonstrated that human corneas after penetrating injuries express high levels of chemokines in limbal epithelium and limbal stroma, moderate levels in corneal stroma, and low expression levels in corneal epithelium. The most striking result in this study was the abundant expression of Gro-α (CXCL-1) and MCP-1 (CCL-2) in the limbus (epithelium more than stroma). Furthermore, the study showed the expression of MIG (CXCL-9), IP-10(CXCL-10), and MIP-1α (CCL-3) in the human cornea, which has, to our knowledge, not been reported before in the literature. Other studies demonstrated the expression of MCP-1 (CCL-2), RANTES (CCL-5), Gro-α (CXCL-1), and IL-8 (CXCL-8) by keratocytes and corneal epithelium.1315 These results are confirmed by other in vivo studies: Hong and colleagues26 demonstrated monocyte chemotactic and activating factor protein in keratocytes of rabbit corneas after epithelial scraping, and Elner and colleagues12 showed the expression of IL-8 in endothelial and stromal cells of human corneas pretreated with IL-1β and tumor necrosis factor α (TNF-α). Taken together, these findings underscore the importance of the limbus and corneal stroma in the immune response of the anterior surface of the eye.

In contrast to the high chemokine expression levels in the limbal epithelium and corneal stroma, several studies have indicated a limited ability of the corneal epithelium to produce chemokines.1215 They reported no expression of MCP-1 (CCL-2) and RANTES (CCL-5) and low expression levels of IL-8 (CXCL-8) in corneal epithelial cells.1215 This study confirms these results. The Gro-α (CXCL-1) was the sole chemokine expressed by the central corneal epithelium. This result, however, is in contrast to recent in vitro studies that failed to detect message for Gro-α (CXCL-1) not only in normal cultures of corneal epithelium but also after stimulation with proinflammatory cytokines TNF-α and IL-1α.14 Although cultured corneal epithelial cells expressed very low levels of IL-8 (CXCL-8)m RNA, they could be significantly stimulated by TNF-α and IL-1α.13 In contrast, we could not detect IL-8 m RNA transcripts in corneal epithelium even in the context of significant inflammation. In our tissue, proinflammatory cytokines needed for the induction of IL-8 in corneal epithelial cells may be missing or a higher expression of this chemokine may occur in earlier or later stages of inflammation. In addition, a selective activation mechanism for high Gro-α m RNA expression or inhibiting factors for IL-8 m RNA expression have to be taken into consideration.

Chemokine studies in our laboratory of inflammatory skin diseases show very similar expression patterns between the skin and the limbus.2731 This study demonstrated the expression of MCP-1 (CCL-2) by the basal limbal epithelium and the expression of Gro-α (CXCL-1) by suprabasal epithelial layers. In skin lesions of psoriasis, lichen planus, and erythema multiforme, MCP-1(CCL-2) is expressed by the basal epithelium, 27,28,31 whereas Gro-α (CXCL-1) is expressed in clusters of keratinocytes.28 An explanation for the different expression patterns of Gro-α (CXCL-1) and MCP-1 (CCL-2) in the limbus might be that proliferating basal epithelial cells produce cytokines distinct from the postmitotic suprabasal cells.

In addition to their differential spatial distribution, studies have demonstrated that chemokines are expressed in a time-dependent manner.24 The study demonstrated that a broad array of chemokines are differentially expressed in inflamed corneas enucleated after a mean of 2 to 4 days. The neutrophil attractant chemokines Gro-α (CXCL-1) and IL-8 (CXCL-8) showed higher expression levels than the mononuclear cell attractant chemokines, MIG (CXCL-9), IP-10(CXCL-10), RANTES (CCL-5), and MIP-1α (CCL-3). These findings are confirmed by wound healing studies performed by our laboratory.32 With the use of a skin repair model in adult humans, Gro-α (CXCL-1), IL-8(CXCL-8), and MCP-1 (CCL-2) are maximally expressed and are associated with a neutrophil infiltration in the first 3 days. From day 4 on, MIG (CXCL-9) and IP-10 (CXCL-10) are highly expressed and paralleled by a mononuclear cell infiltration. It is, therefore, reasonable to suggest that, in the further time course of corneal inflammation, mononuclear cell attractant chemokines such as IP-10 (CXCL-10) and MIG (CXCL-9) will be up-regulated. This notion is confirmed by chemokine studies by our group on specimens with squamous cell carcinoma of the conjunctiva associated with chronic inflammation. The conjunctival epithelium showed foci with high expression levels of MIG (CXCL-9) in accordance with a high presence of mononuclear cells in the subepithelial epithelium (U.H.M.S., A.T., S.V., and R.G., unpublished results, 1999).

In conclusion, the differential chemokine production in limbus and cornea(limbus greater than corneal stroma greater than corneal epithelium) may be necessary to attract specific leukocyte subsets to different layers of the ocular surface.1215 The corneal epithelium secretes significant levels of Gro-α (CXCL-1), to initiate an acute inflammatory response directed against superficial infections controllable by neutrophils. If the infection spreads to the corneal stroma, a more intense and longer-lasting immune reaction is needed to attract mononuclear cells to the cornea. Therefore, the expression of mononuclear cell attractant chemokines such as MCP-1 (CCL-2), MIP-1α (CCL-3), and MIG (CXCL-9) by corneal keratocytes, as shown in this study in vivo and other studies in vitro, 1215 may be triggered despite the risk of vision loss to protect the content of the eye.2631

Experimental studies on inflamed corneas open several possible therapeutic options for their clinical management.1215,26 The expression of proinflammatory cytokines in the cornea such as IL-1α and TNF-α has been demonstrated in response to trauma such as epithelial scrape injury, 33 alkalide burn, 34 and UV irradiation.35 Several clinical studies report a significant reduction of inflammation in rheumatoid arthritis and Behçet disease after treatment with anti–TNF-α and anti–IL-1α.3638 The inhibition of proinflammatory cytokines would therefore be a reasonable strategy to limit the infiltration of leukocytes into corneal tissue. In addition, corticosteroids such as betamethasone inhibit cell infiltration into the aqueous humor and IL-8 expression in an endotoxin-induced uveitis rat model.39 The selective inhibition of a leukocyte subset, however, can only be achieved by means of selective chemokine antibodies such as anti–IL-8.12

Further in vivo investigations are needed to demonstrate the expression of proinflammatory cytokines and chemokines in different inflammatory eye diseases. Selective receptor blockage of highly expressed chemokines or proinflammatory cytokines in inflamed eye tissue could then be explored as a reasonable strategy to inhibit the recruitment of leukocytes.

Corresponding author and reprints: Ulrich H. M. Spandau, MD, Department of Ophthalmology, University of Heidelberg, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany (e-mail: Ulrich.Spandau@augen.ma.uni-heidelberg.de).

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Submitted for publication May 14, 2002; final revision received January 9, 2003; accepted February 12, 2003.

References
1.
Chandler  JWGillette  TE Immunologic defense mechanisms of the ocular surface. Ophthalmology. 1983;90585- 591Article
2.
Nelson  PJKrensky  AM Chemokines, chemokine receptors, and allograft rejection. Immunity. 2001;14377- 386Article
3.
Rossi  DZlotnik  A The biology of chemokines and their receptors. Annu Rev Immunol. 2000;18217- 242Article
4.
Luster  AD Chemokines--chemotactic cytokines that mediate inflammation. N Engl J Med. 1998;12436- 445
5.
Larson  CGAndersen  AOApella  EOppenheim  JJMatsushima  K The neutrophil activating protein (NAP-1) is also chemotactic for T-lymphocytes. Science. 1989;2431464- 1466Article
6.
Jinquan  TFrydengerg  JMukaida  N  et al.  Recombinant human growth-regulated oncogene-α induces T lymphocyte chemotaxis. J Immunol. 1995;1555359- 5368
7.
Loetscher  MGerber  BLoetscher  P  et al.  Chemokine receptor specific for IP-10 and MIG: structure, function and expression in activated T-lymphocytes. J Exp Med. 1996;184963- 969Article
8.
Schall  TJBacon  KToy  KJGoeddel  DV Selective attraction of monocytes and T-lymphocytes of the memory phenotype by cytokine RANTES. Nature. 1990;347669- 671Article
9.
Taub  DDConlon  KLloyd  AROppenheim  JJKelvin  DJ Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1α and MIP-1β. Science. 1993;260355- 358Article
10.
Carr  MWRoth  SJLuther  ERose  SSSpringer  TA MCP-1 acts as a T-lymphocyte chemoattractant. Proc Natl Acad Sci U S A. 1994;913652- 3656Article
11.
Uguccioni  MD'Apuzzo  MLoetscher  MDewald  BBaggiolini  M Actions of the chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 α and MIP-1 β on human monocytes. Eur J Immunol. 1995;2564- 68Article
12.
Elner  VMStrieter  RMPavilack  MA  et al.  Human corneal interleukin-8, IL-1 and TNF-induced gene expression and secretion. Am J Pathol. 1991;139977- 988
13.
Cubitt  CLTang  QMonteiro  CALausch  RNOakes  JE IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes. Invest Ophthalmol Vis Sci. 1993;343199- 3206
14.
Cubitt  CLLausch  RNOakes  JE Differential induction of GRO-α gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines. Invest Ophthalmol Vis Sci. 1997;381149- 1158
15.
Tran  MTTellaetxe-Isusi  MElner  VStrieter  RMLausch  RNOakes  JE Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. Invest Ophthalmol Vis Sci. 1996;37987- 996
16.
Farber  JM Hu MIG: a new member of the chemokine family of cytokines. Biochem Biophys Res Commun. 1993;192223- 230Article
17.
Liao  FRabin  RLYanelli  JRKoniaris  LGVanguri  PFarber  JM Human Mig chemokine: biochemical and functional characterization. J Exp Med. 1995;1821301- 1314Article
18.
Luster  ADRavetch  JV Biochemical characterization of a γ-interferon inducible cytokine(IP-10). J Exp Med. 1987;1661084- 1097Article
19.
Vanguri  PFarber  JM Identification of CRG-2: an interferon inducible m RNA predicted to encode a murine monokine. J Biol Chem. 1990;26515049- 15057
20.
Anisowicz  ABardwell  LSager  R Constitutive overexpression of a GROwth-regulated gene in transformed Chinese hamster and human cells. Proc Natl Acad Sci U S A. 1987;847188- 7192Article
21.
Richmond  ABalentien  EThomas  HG  et al.  Molecular characterization and chromosomal mapping of melanoma GROwth stimulatory activity, a GROwth factor structurally related to β-thromboglobulin. EMBO J. 1988;72025- 2033
22.
Yoshimura  TMatsushima  KTanaka  S  et al.  Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc Natl Acad Sci U S A. 1987;849233- 9237Article
23.
Yoshimura  TYuhki  NMoore  SKAppella  ELerman  MILeonard  EJ Human monocyte chemoattractant protein-1 (MCP-1): full-length c DNAcloning, expression in mitogen-stimulated blood mononuclear leukocytes and sequence similarity to mouse competence gene JE. FEBS Lett. 1989;244487- 493Article
24.
Schall  TJJongstra  JDyer  B  et al.  Human T-cell specific molecule is a member of a new gene family. J Immunol. 1988;1411018- 1025
25.
Wolpe  SDDavatelis  GSherry  B  et al.  Macrophages secrete a novel heparin binding protein with inflammatory and neutrophil chemokinetic properties. J Exp Med. 1988;167570- 581Article
26.
Hong  JWLiu  JJLee  JS  et al.  Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci. 2001;422795- 2803
27.
Gillitzer  RWolff  KTong  D  et al.  MCP-1 m RNA expression in basal keratinocytes of psoriatic lesions. J Invest Dermatol. 1993;101127- 131Article
28.
Gillitzer  RRitter  USpandau  UGoebeler  MBröcker  EB Differential expression of GRO-α and IL-8 m RNA in psoriasis: a model for neutrophil migration and accumulation in vivo. J Invest Dermatol. 1996;107778- 782Article
29.
Goebeler  MToksoy  ASpandau  UEngelhardt  EBröcker  EBGillitzer  R The CXC chemokine MIG is highly expressed in the papillae of psoriatic lesions. J Pathol. 1998;18489- 95Article
30.
Spandau  UToksoy  AGoebeler  MGillitzer  R MIG is a dominant lymphocyte-attractant chemokine in lichen planus lesions. J Invest Dermatol. 1998;1111003- 1009Article
31.
Spandau  UBröcker  EBKämpgen  EGillitzer  R C-C and C-X-C chemokines are differentially expressed in erythema multiforme in vivo. Arch Dermatol. 2002;1381027- 1033Article
32.
Engelhardt  EToksoy  AGoebeler  MDebus  SBroecker  EBGillitzer  R Chemokines IL-8, Gro-α, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am J Pathol. 1998;1531849- 1860Article
33.
Wilson  SEHe  YGWeng  J  et al.  Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res. 1996;62325- 327Article
34.
Planck  SRRich  LFAnsel  JCHuang  XNRosenbaum  JT Trauma and alkali burns induce distinct patterns of cytokine gene expression in the rat cornea. Ocul Immunol Inflamm. 1997;595- 100Article
35.
Kennedy  MKim  KHHarten  B  et al.  Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci. 1997;382483- 2491
36.
Taylor  PC Anti-TNF therapy for rheumatoid arthritis and other inflammatory diseases. Mol Biotechnol. 2001;19153- 168Article
37.
Sfikakis  PP Behcet's disease: a new target for anti-tumour necrosis factor treatment. Ann Rheum Dis. 2002;61 ((suppl 2)) II51- II53
38.
Arend  WP The mode of action of cytokine inhibitors. J Rheumatol Suppl. 2002;6516- 21
39.
Tsuji  FSawa  KKato  MMibu  HShirasawa  E The effects of betamethasone derivatives on endotoxin-induced uveitis in rats. Exp Eye Res. 1997;6431- 36Article
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