Published online before print November 16, 2006
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Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio, USA
1 Correspondence: Department of Ophthalmology and Center for Global Health and Diseases, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-7286, USA. E-mail: eric.pearlman{at}case.edu
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Key Words: chemokine • macrophage • inflammation • MIP-2
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The chemokine family is divided into four main groups based on their structure and chemotactic activity for specific leukocyte populations: C, CC, CXC, and CX3C. The subset of CXC chemokines containing a glycine-leucine-arginine (ELR) motif, immediately preceding the CXC residues, selectively target neutrophils. Although there are seven ELR+ CXC chemokines in the human genome, only four have been identified in the murine genome: CXCL1/keratinocyte-derived chemokine (KC), CXCL2/monocyte-inhibitory protein-2 (MIP-2), CXCL5/LPS-induced chemokine (LIX), and CXCL15/lungkine [8 9 10 11 12 ]. KC was first identified from a cDNA library of a platelet-derived growth factor-induced fibroblast line [12 ]. MIP-2 was initially isolated from a LPS-stimulated macrophage cell line, RAW 264.7 [9 ]. LIX, originally identified from LPS-stimulated fibroblasts, was shown to have a role in neutrophil recruitment in rodent models of cardiac ischemia-reperfusion, colitis, and LPS-induced acute lung inflammation [8 ,10 ,13 14 15 16 ]. Although ELR+ CXC chemokines mediate the regulation of neutrophil recruitment, migration, and activation at sites of infection/injury, the relative contribution of each CXC chemokine to these functions remains unclear.
To elucidate the role of these chemokines in neutrophil recruitment into the cornea, we examined the kinetics of CXC chemokine production in a mouse model of LPS-induced keratitis [17 ,18 ] and also examined the production of these chemokines in corneal fibroblasts and peritoneal macrophages and neutrophils. In addition, we used neutralizing antibody to determine the role of these chemokines in corneal inflammation.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Murine model of LPS keratitis
Four- to 8-week-old female C57BL/6 mice were anesthetized by i.p. injection with 0.4 ml of a 1.2% solution of 2,2,2-tribromoethanol (Sigma-Aldrich) containing 2.5% 2-methyl-2-butanol (tertiary amyl alcohol, Sigma-Aldrich) dissolved in distilled water. After creation of a tunnel into the corneal stroma using a 33-gauge needle, 1 µg LPS in 2 µl HBSS (Invitrogen, Carlsbad, CA, USA) was injected into the central corneal stroma via a separate 33-gauge needle attached to a 5-µl glass syringe (Hamilton Co., Reno, NV, USA) as described previously [17
]. Control corneas were injected with 2 µl HBSS. All mice were treated in accordance with The Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Histology and immunohistochemistry
Mice were killed, and eyes were enucleated and snap-frozen in OCT (Tissue-Tek, Sakura, Torrance, CA, USA) using liquid nitrogen. Cryosections (5 µm) were stained for neutrophils using the rat antimouse mAb NIMP-R14, which is specific for neutrophils [20
]. Briefly, sections were fixed in 4% formaldehyde for 30 min and rinsed in 0.05 M Tris buffer solution, pH 7.6. Sections were then incubated with the 8-µg/ml NIMP-R14 for 2 h. After washing, sections were incubated for 45 min with 5 µg/ml FITC-conjugated antirat IgG (H+L, Vector Laboratories, Burlingame, CA, USA). Sections were mounted with VectaShield mounting medium containing 4',6-diamidino-2-phenylindole (Vector Laboratories). Neutrophils, per 5 µm section, were counted (from limbus to limbus at 400x) by fluorescence microscopy (Olympus Optical Co. Ltd., Tokyo, Japan).
Cell culture and activation
MK/T-1 cells were maintained in DMEM/low glucose containing 10% FBS and 50 µg/ml hygromycin (Invitrogen) at 37°C with 5% CO2. Cells were then stimulated with 100 ng/ml LPS in DMEM without hygromycin and FBS. For controls, cells were incubated with media alone. Supernatants collected at the indicated time-points were analyzed for KC, MIP-2, and LIX by ELISA.
Neutrophil isolation and activation
Neutrophils were isolated from the peritoneal cavity as described previously [21
,22
]. Mice were injected twice i.p. with 1% casein solution containing 0.5 mM MgCl2 and 0.9 mM CaCl2. Three hours after the second casein injection, a peritoneal lavage with 10 ml HBSS was performed to collect cells. After washing with HBSS, a cell count was performed using trypan blue, and the cells were brought to a concentration of 3–5 x 107 cells/ml in 1x PBS. Cell solution (1 ml) was added to a Percoll gradient solution (GE Healthcare Biosciences) and centrifuged at 31,500 rpm in an ultracentrifuge (Beckman-Coulter, Fullerton, CA, USA) with a 50-Ti rotor for 20 min at 4°C. The neutrophil cell layer was collected, washed with HBSS, and resuspended in DMEM containing 10% FBS. Diff-Quik stain (VWR, Bridgeport, NJ, USA) showed >98% neutrophil purity. Cells (1x105 per well) were preincubated in GM-CSF for 18 h at 37°C, with or without LPS (100 ng/ml). Supernatants were collected and assayed for the presence of chemokines by ELISA.
Macrophage isolation and activation
Macrophages were isolated from the peritoneal cavity using casein. Briefly, mice were injected i.p. with 1% casein solution containing 0.5 mM MgCl2 and 0.9 mM CaCl2. After 24 h, a peritoneal lavage with 10 ml HBSS was performed to collect cells. After lysis of RBC using RBC lysis buffer (Sigma-Aldrich), remaining cells were washed with HBSS. A cell count was performed using trypan blue, and cells were resuspended in DMEM containing FBS, penicillin, and streptomycin (DMEM/FBS/P/S). Cells were allowed to adhere to the tissue-culture plate for 2 h. After removal of adherent cells, cells were incubated overnight at 37°C, 5% CO2, in DMEM/FBS/P/S. Cells were then incubated with or without 100 ng/ml LPS in DMEM alone. Supernatants were collected at indicated time-points and assayed for the presence of chemokines by ELISA.
Quantification of CXC chemokines by ELISA
After LPS or HBSS treatment as indicated above, corneas were excised at the indicated time-points, the epithelium was removed following an incubation in 20 mM EDTA for 20 min, and the stroma were suspended in 200 µL RPMI 1640. As described previously, samples were sonicated for 88 s with 50% duty cycle (VibraCell, Sonics and Materials, Danbury, CT, USA) [6
]. Tissue and in vitro LPS-stimulated, cell-free supernatant chemokine levels (collected as described above) were assessed by a sandwich ELISA according to the manufacturer’s protocol (R&D Systems). The limit of detection of the assays was 15 pg/ml.
Generation of enhanced GFP (eGFP) chimeric mice
eGFP chimeric mice were prepared as described previously [17
]. Briefly, isolated bone marrow from the femur and tibia of C57Bl/6-transgenic mice (ACTbGFP, Jackson Laboratories) was reconstituted in sterile HBSS, incubated briefly in RBC lysis buffer (eBioscience, San Diego, CA, USA), and resuspended in DMEM. Bone marrow cells (2–5x106) were injected i.v. (tail-vein) in 0.2 ml into recipient C57Bl/6 mice, which had been exposed to 2 x 600 
-doses of whole-body radiation 3 h apart. The eGFP chimeric mice were then used for subsequent experiments 2 weeks later.
In vivo inhibition of eGFP inflammatory cells
Corneas of eGFP chimeric mice were injected with 2 ul containing 15 ug neutralizing antibody to KC, LIX, MIP-2, control rat IgG2a, or control rat IgG2b 2 h prior to intrastromal injection of 200 ng LPS in 0.5 µl HBSS. [The anti-MIP-2 antibody 50% neutralizing dose (ND50) is 1–4 ug/ml for 2 ng/ml recombinant murine (rm)MIP-2; anti-KC ND50, 1–4 ug/ml antibody for 0.5 ug/ml rmKC; and anti-LIX ND50, 0.5–2.5 ug/ml antibody for 0.2 ug/ml rmLIX.] Twenty hours after LPS injection, in vivo fluorescence stereomicroscopic images were captured using a MZFLIII microscope and Spot RT Slider KE camera (Leica Microsystems Inc., Bannockburn, IL; Diagnostics Instruments, Sterling Heights, MI, USA) as described previously [17
]. The number of eGFP cells was quantified in a constant-defined area of the image (ImagePro Plus, Media Cybernetics, Carlsbad, CA, USA). Mice were killed, eyes were enucleated, and neutrophils were assessed as described above. Difference in the means was calculated, and percent inhibition was derived using Prism GraphPad software (San Diego, CA, USA).
Statistical analysis
Statistical analysis was performed with an unpaired t-test (Prism, GraphPad Software). P < 0.05 was considered significant.
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As shown in Figure 1
, neutrophils were detected as early as 3 h after LPS injection and were mostly in the peripheral cornea (not shown). By 8 h, neutrophil numbers had increased and were detected throughout the cornea. Total neutrophil numbers peaked at 24 h and declined by 48 h. For CXC production, KC levels increased 800-fold within 3 h of injection and continued to increase to 
1500 pg/ml at 24 h. After this time, KC levels decreased in parallel with neutrophil numbers. MIP-2 was also detected within 3 h but remained at 100 pg/ml for 24 h. In contrast, LIX was not detected until 8 h postinjection and gradually increased to 500 pg/ml at 24 h. By 48 h, KC and LIX levels had decreased and remained low or undetectable. In this model of corneal inflammation, chemokine production by naïve corneas and by corneas injected with saline was below the limit of detection (not shown).
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Figure 1. Neutrophil infiltration and CXC chemokine production in the corneal stroma during LPS keratitis. Corneas were injected intrastromally with 1 ug LPS. After 0, 3, 8, 24, 48, and 72 h, the corneas were sonicated, and KC ( ), LIX (•), and MIP-2 ( ) levels were determined by ELISA. Limit of detection was 15 pg/ml. At the indicated time-points, additional eyes were enucleated, sectioned, and immunostained with anti-NIMP-R14. The number of neutrophils (solid line) was determined by direct counting in 5 µm sections. Results are mean ± SEM of six individual corneas per time-point and are representative of four experiments.
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As shown in Figure 2 , LPS-stimulated corneal stromal fibroblasts produced LIX and KC with distinct temporal patterns. KC was detected within 2 h after stimulation and peaked within 6 h, whereas LIX production increased gradually, reaching similar concentrations after 18 h. MIP-2 was not detected (<15 pg/ml), even after 18 h. These findings demonstrate clearly that corneal fibroblasts produce KC and LIX but not MIP-2 in response to LPS treatment.
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Figure 2. Production of KC, LIX, and MIP-2 by corneal fibroblasts, neutrophils, and macrophages after LPS stimulation. MK/T-1 corneal fibroblasts, peritoneal neutrophils, and macrophages were stimulated with 100 ng/ml E. coli LPS. At each time-point, supernatants were collected and analyzed for the presence of KC (), LIX (•), and MIP-2 () by ELISA. Limit of detection was 15 pg/ml. The results are representative of one of three experiments performed in triplicate (mean±SEM).
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Taken together, these results demonstrate a distinct cellular pattern of CXC chemokine production, where corneal fibroblasts are a major source of KC and LIX, but not MIP-2, and neutrophils and macrophages are a major source of MIP-2 and a minor source of KC but do not produce LIX.
CXCL1/KC and CXCL5/LIX mediate neutrophil infiltration to the corneal stroma
To determine which CXC chemokines mediate neutrophil recruitment to the cornea and migration through the corneal stroma, neutralizing antibodies to KC, LIX, or MIP-2 were injected into the corneal stroma of eGFP chimeric mice 2 h prior to LPS injection. These conditions are consistent with other studies [17
,18
,33
] and are within the neutralizing capacity of these antibodies as described in Materials and Methods. Infiltration of total eGFP bone marrow-derived cells into the corneal stroma of live mice was detected by fluorescence microscopy, and images of the whole cornea were captured for analysis. After sacrifice, the eyes were then sectioned, and corneas were examined by immunohistochemistry to determine the number of neutrophils in the cornea.
Figure 3A shows representative images of whole corneas, flattened images (background fluorescence equalized), and analysis of cellular infiltrate at 20 h post-LPS injection. Figure 3B shows quantitative analysis of four corneas per group. Corneas of eGFP chimeric mice at 20 h post-LPS injection in the presence of control antibody showed an intense infiltrate of bone marrow eGFP cells throughout the cornea. Infiltration of these cells was significantly lower in corneas injected with anti-LIX or anti-KC (58±19.8% and 64±13.5% reduction, respectively; Fig. 3B ). In contrast, there was no significant difference in the number of bone marrow-derived cells between anti-MIP-2 and control IgG isotype-treated corneas. Figure 3C shows that the number of neutrophils was also decreased significantly in corneas treated with anti-KC (54±5.6%) or anti-LIX (56±16%) compared with IgG isotype control-treated corneas, whereas anti-MIP-2-treated corneas did not show a significant decrease in neutrophil infiltration.
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Figure 3. Effect of KC, LIX, or MIP-2 neutralization on eGFP cellular and neutrophil infiltration in the cornea. Antibodies to KC, LIX, and MIP-2 or isotype controls were injected in corneas of eGFP chimeric mice. After 1 h, LPS (200 ng) was injected into the corneal stroma, and eyes were examined by fluorescence microscopy after 20 h. (A) Left panel shows representative in vivo fluorescence images (25x magnification; 3 s exposure) of eGFP-positive cells in corneas after 20 h; middle panel depicts flattened image with decrease background fluorescence; and right panel shows quantification of eGFP-positive cells (in red) in a defined region on the image. (B) Mean ± SEM of eGFP cells per group (n=4). (C) Mean ± SEM of neutrophils per group (n=4). These data are representative of two experiments.
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Figure 4. Effect of combined KC, LIX, and MIP-2 neutralization on eGFP cellular and neutrophil infiltration in the cornea. LPS (200 ng) was injected into the corneal stroma of eGFP chimeric mice 2 h following injection of isotype IgG control, a combination of anti-KC and anti-LIX, or a combination of anti-KC, anti-LIX, and anti-MIP-2 mAb (all at the same final concentration). (A) eGFP cells and neutrophils in corneas given anti-KC and anti-LIX or control IgG antibody treatment. (B) eGFP cells and neutrophils in the corneas given anti-KC, anti-LIX, and anti-MIP-2 or control IgG antibody treatment. Data are representative of one of two experiments with four corneas per group (mean±SEM).
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, we focused on the murine ELR+ chemokines CXCL1/KC, CXCL5/LIX, and CXCL2/MIP-2, which bind CXCR2 on neutrophils. Our findings demonstrate that chemokine expression occurs in a temporal pattern; KC is expressed prior to neutrophil infiltration and at high levels, and LIX production is detected later and at lower levels (threefold less than KC) but parallels neutrophil infiltration to the corneal stroma.
In addition to the temporal pattern of chemokine expression, we observed cell-specific expression of CXC chemokines. Whether resident keratocytes express TLR4 or respond to LPS is uncertain; however, keratocytes differentiate into fibroblasts under inflammatory conditions and can produce a number of cytokines in response to LPS [23 24 25 26 27 28 ]. Similarly, the MK/T-1 stromal fibroblast line used in the current study responds to highly purified LPS. The corneal fibroblasts in culture selectively express KC and LIX, and neutrophils produce MIP-2 and KC. Based on this differential production of chemokines, we anticipate that the KC and LIX detected in vivo originate from corneal fibroblasts. This is consistent with a mouse model of surgical injury in the skin in which KC is expressed primarily in dermal fibroblasts and endothelial cells, whereas MIP-2 is restricted to infiltrating leukocytes [35 ]. Similarly, a rat model of ischemia-reperfusion showed that LIX was expressed by resident myocardial cells, whereas MIP-2 localized primarily to infiltrating cells [14 ]. Our findings that LIX is produced selectively by corneal fibroblasts and that infiltrating neutrophils and macrophages serve as the predominant source of MIP-2 support Hamilton’s proposal that there is a nonmyeloid and myeloid pattern of chemokine production and also introduce the possibility that LIX is a "nonmyeloid" chemokine [35 ]. Further studies remain to determine whether LIX is produced exclusively by nonmyeloid cells. These findings are distinct from a report where G-CSF-stimulated human neutrophils increase mRNA expression of CXCL5/epithelial-derived neutrophil-activating factor-78 (ENA-78), a human homologue of LIX, and anti-ENA-78 antibodies abrogate neutrophil chemotaxis [36 ]. Although the argument remains that murine neutrophils could produce LIX under certain stimulatory conditions, the same study found no expression of CXCL6/granulocyte chemotactic protein-2 (GCP-2) from stimulated or unstimulated human neutrophils. Although LIX shares 55% sequence homology to human CXCL5/ENA-78, it also shares 61% homology to human CXCL6/GCP-2 and is therefore no more closely related to ENA-78 than to GCP-2 [37 ]. In contrast to LIX, we showed that corneal fibroblasts did not produce MIP-2 in vitro, although in vivo results show the detection of MIP-2 as early as 3 h. Bone marrow-derived cells are present in the normal murine cornea, and activated macrophages in vitro produce MIP-2, as we and others have shown [38 39 40 ]. Therefore, the MIP-2, detected prior to recruitment of neutrophils, likely originates from resident macrophages. Once present in the corneal stroma, release of prestored MIP-2 from neutrophils likely contributes to its production in the cornea [29 ].
Although all of these chemokines bind CXCR2, the cell-specific production of CXC chemokines and their temporal expression could indicate a complementary network of chemokine function in the cornea. Based on these results, we propose a sequence of events regarding the chemokine network and its contribution to neutrophil infiltration and inflammation: Upon interaction with LPS, resident stromal cells and macrophages produce KC and MIP-2. These chemokines, in turn, recruit neutrophils from peripheral vessels into the corneal stroma. Once present in the corneal stroma and exposed to LPS, neutrophils release MIP-2 to continue the cycle of neutrophil recruitment and chemotaxis. Simultaneously, the corneal fibroblasts can up-regulate LIX and KC production to further mediate recruitment of neutrophils into the inflamed cornea. Although this sequence of events is speculative, we tested in vivo the biological role of KC, LIX, and MIP-2 in neutrophil recruitment by treating corneas with neutralizing antibodies prior to LPS challenge. Use of anti-LIX or anti-KC antibodies significantly inhibited neutrophil recruitment to the cornea, thereby identifying a functional role for these chemokines in LPS keratitis. Moreover, our data show that only corneal fibroblasts produce LIX, indicating that these cells play a direct role in neutrophil infiltration. Other models of corneal inflammation and wound-healing have shown production of KC and MIP-2 and more recently, LIX in the cornea, but the role of LIX in mediating neutrophil infiltration to the cornea has not been examined [41
42
43
]. In the present study, CXCL1/KC is the predominant CXC chemokine produced, and neutralization significantly inhibits recruitment of neutrophils to the corneal stroma. Xue et al. also observed that KC neutralization decreased neutrophil emigration in P. aeruginosa-induced keratitis [44
]. Their study and others also showed a role for MIP-2 in infiltration of neutrophils to the cornea in models of microbial keratitis [17
,18
,33
,45
]. In contrast, we found that although MIP-2 is produced, local administration of anti-MIP-2, alone or in combination with anti-KC and anti-LIX, had no significant effect on neutrophil recruitment. The absence of significant neutralizing activity was not a result of lack of antibody function, as we found that it bound MIP-2 in vitro (data not shown). The difference between these reports and our findings has yet to be determined but may relate to the initial wave of neutrophil recruitment to the corneal stroma versus migration through the corneal stroma. Although we also found that simultaneous injection of antibodies to KC, LIX, and MIP-2 did not completely inhibit neutrophil recruitment and migration to the cornea, it is possible that additional neutrophil chemoattractants, including monocyte-inhibitory protein-1
(MIP-1), acetylated-proline glycine-proline (acPGP)-containing collagen fragments generated during the inflammatory process, or complement mediators such as C3a and C5a may contribute to recruitment [46
47
48
].
Our study focuses on the role of chemokines in neutrophil migration; however, ELR+ CXC chemokines have differential effects on neutrophils and can regulate other cells, although they all signal through CXCR2. These functions include non-neutrophil-related functions such as angiogenesis and monocyte arrest [43
,49
,50
]. Studies indicating that these chemokines can have distinct functions contributing to neutrophil infiltration as well as the inflammatory response include elastase release, NF-
B activation, integrin expression, selectin shedding, and selective transport into the systemic circulation [51
52
53
54
55
]. Although these mechanisms have not been elucidated in the cornea, results from the present study clearly demonstrate an important role for KC and LIX in mediating neutrophil recruitment and infiltration to the corneal stroma and may provide the basis for a more effective strategy for the treatment of bacterial keratitis.
Received August 3, 2006; revised September 29, 2006; accepted October 22, 2006.
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regulates platelet endothelial cell adhesion molecule 1 expression and neutrophil infiltration into herpes simplex virus-infected mouse corneas J. Exp. Med. 184,1435-1447B and induces neutrophil infiltration via lipopolysaccharide-induced CXC chemokine Circulation 103,2296-2302-defensin-3 via CpG-ODN-TLR-9 in corneal fibroblasts Br. J. Ophthalmol. 90,378-382 prevents the development of blinding herpes stromal keratitis J. Virol. 72,3705-3710 B pathway J. Biol. Chem. 278,4675-4686This article has been cited by other articles:
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