Published online before print September 22, 2006
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* Department of Microbiology and Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee, USA; and
Department of Pediatric Infectious Disease, Vanderbilt University, Nashville, Tennessee, USA
2 Correspondence: M409 Walters Life Sciences Building, University of Tennessee, Knoxville, TN, 37996-0845, USA. E-mail: btr{at}utk.edu
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Key Words: chemokine • Herpes simplex virus • cornea
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, MIP-1 β, RANTES [4
, 5
], and the accumulation of innate inflammatory cells, including neutrophils and macrophages [3
, 6
]. Once at the site, activated inflammatory cells produce various proinflammatory cytokines [7
], proteinases [8
], and angiogenic factors [9
], which cause further tissue damage. It is likely that several chemoattractants are key factors in the pathogenesis of SK and that an interdependent network between these molecules may be involved in the disease process. For example, the overexpression or down-regulation of one molecule may influence the expression of many other related or unrelated molecules. However, the mechanisms by which the balance between inflammatory molecules is controlled in different disease models are still undefined. In some previous studies, HSV ocular infection was shown to result in the enhanced expression of several chemokines [4 , 10 ], but the function of individual molecules is not well understood. The chemokine monocyte chemoattractant protein-1 (MCP-1) is known to play a pivotal role in several pathological conditions, including acute toxoplasmosis, granuloma formation, and lethal endotoxemia [11 12 13 ], and this molecule is up-regulated during HSV ocular infection [10 ]. MCP-1 acts as a chemoattractant factor for immune cells, mainly monocyte/macrophage, neutrophils, and activated T cells in pathological, as well as physiological conditions [14 , 15 ]. Furthermore, MCP-1 was shown to increase disease severity in a model of HSV-2-induced encephalomyelitis by stimulating Th-2 responses [16 ]. Thus MCP-1 could represent a pivotal chemokine in SK. However, MCP-1 function appears redundant and can be replaced by the activity of other chemokines [17 ]. Accordingly, many CC or CXC chemokines have chemoattractant activity against inflammatory cells, and different cell types may express different kinds of chemokine receptors [18 ]. In consequence, lacking a single chemokine may or may not be expected to have a dramatic phenotype. However, because MCP-1 was found to be highly expressed after HSV infection of the cornea [10 ], the effect on lesion development in mice lacking MCP-1 was investigated. Our results show that the loss of MCP-1 resulted in more severe angiogenesis and SK lesions. This result appeared to be the consequence of overexpression of MIP-2 or other chemokines and increased infiltration of inflammatory cells expressing Gr-1 and CD11b into the SK cornea.
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Mice
Female BALB/c mice (H-2d), 6 to 7 weeks old, were purchased from Harlan Sprague-Dawley (Indianapolis, IN). MCP-1–/– mice in a BALB/c background were kindly provided by Dr. B. J. Rollins (Dana-Faber Cancer Institute, Harvard Medical School, Boston, MA). MCP-1–/– strain was derived in BALB/c-129Sv/J F1 and backcrossed against BALB/c mice for 10 generations. Thus, we used BALB/c mice as controls in experiments with MCP-1–/– mice. All investigations followed guidelines of the Committee on the Care of Laboratory Animals Resources, Commission of Life Sciences, National Research Council. The animal facilities of the University of Tennessee (Knoxville) are fully accredited by the American Association of Laboratory Animal Care.
Virus
HSV-l RE strain (obtained from the laboratory of R. Hendricks, University of Pittsburgh, Pittsburgh, PA, USA) was used in all procedures. Virus was grown in Vero cell monolayers (American Type Culture Collection, Manassas, VA, USA; Cat. no. CCL81), titrated, and stored in aliquots at –80°C until used.
Corneal HSV infection
Corneal infections of all mouse groups were conducted under Avertin deep anesthesia. The mice were scarified lightly on their corneas with a 30-gauge needle, and a 2-µl drop containing 1 x 105 plaque-forming units (PFU) of HSV-1 RE was applied to the eye and gently massaged with the eyelids.
cDNA array analysis
At days 1, 3, 5 after ocular infection, the corneas were isolated, and four at each time point were immediately excised and transferred to Tri-reagent (Molecular Biology, Cincinnati, OH). The total RNA was extracted according to the manufacturer’s directions. Aliquots of RNA (10 µg) were reverse transcribed into cDNA. The resulting cDNA probes were hybridized to mouse gene specific for angiogenic factors that were spotted on the GEArray membranes (SuperArray Inc., Bethesda, MD). The relative amount of a given gene transcript was estimated by comparing its signal intensity with the signal derived from GAPDH and β-actin on the same membrane. A more than threefold increase in signal intensity was considered significant.
RNA extraction and RT-PCR
At various time points after ocular infection, the corneas were isolated, and two at each time point were immediately excised and transferred to Tri-reagent (Molecular Biology). Total RNA from cells was extracted by using RNeasy RNA extraction kit (Qiagen, Valencia, CA). Briefly, cells were lysed in lysis buffer, and RNA was purified following the manufacturer’s protocol. DNase treatment (Qiagen) was done to remove genomic DNA. Total cellular RNA (10 µg/ml) was reversed-transcribed using oligo (dT) primers and RT (Promega, Madison, WI), according to protocols described previously [19
]. The cDNA was made by the reverse-transcription reaction incubated at 42°C for 90 min. RT-PCR was performed according to the manufacturer’s protocol (HotStar taq master mix kit, Qiagen) with 2 µg of total RNA. The following RT-PCR conditions were used: 1 cycle of 50°C for 5 min followed by 1 cycle of 94°C for 1 min: 30 cycles of 94°C for 1 min, 56°C for 1 min and 72°C for 1 min and a final cycle of 72°C for 10 min. The primer sequences for MCP-1 were 5'-GCCAGACGGGAGGAAGGC-3' (sense) and 5'-GGCATCACAGTCCGAG-3' (antisense). RT-PCR products were 546 bp. The PCR products were separated by 1% agarose gel electrophoresis.
Protein quantification of corneal lysates by ELISA
The lysates from CpG ODNs-implanted, as well as HSV-infected, cornea were used for the measurement of MCP-1, MIP-2, MIP-1, vascular endothelial growth factor (VEGF), or IL-1β by a standard sandwich ELISA protocol. For preparation of corneal lysates, four corneas/time point (n = 4) were collected and minced with liquid nitrogen. Minced pieces were collected in 1 ml of DMEM without FCS and homogenized using an ultra sonicater (Heat Systems-Ultrasonics, Plainview, NY). The lysates were then clarified by centrifugation at 12,000 rpm for 5 min at 4°C. The supernatant was collected and stored at –80°C until further use. The ELISA plate was coated with anti-mouse capture antibody (100 µl/well of the capture antibody at various concentrations, R&D Systems, Minneapolis, MN) and incubated at 4°C overnight. The plate was washed with 0.05% Tween 20/PBS and blocked with 3% BSA for 2 h at 37°C. After washing, serially diluted corneal lysates were added to the plate and incubated at 4°C overnight. The plate was washed and then incubated with biotinylated detection antibody (R&D Systems) for 2 h. Finally, peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratory, West Grove, PA) was added. The color reaction was developed using ABTS (Sigma-Aldrich, St. Louis, MO) and measured with an ELISA reader (Spectramax 340; Molecular Devices, Sunnyvale, CA) at 405 nm. Quantification was performed with Spectramax ELISA reader software version 1.2.
Immunohistochemistry
At day 2 after infection, eyes were removed and snap-frozen in OCT compound (Miles Inc., Elkhart, IN). Sections (6 µm thick) were cut, air-dried, and fixed in cold acetone for 10 min. The sections were then blocked with 3% BSA and stained with anti–mMCP-1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h, which were followed by incubation with biotinylated anti–goat Ig (Santa Cruz Biotechnology) for 1 h. The sections were then incubated with horseradish peroxidase–conjugated streptavidin (1:1,000; Jackson Immunoresearch Laboratories) for 1 h, and color reactions were developed with 3,3'-diaminobenzidine substrate (BioGenex Laboratories, San Ramon, CA).
Clinical observations
The eyes were examined on different days after infection for the development of clinical lesions by slit-lamp biomicroscopy (Kawa Co., Nagoya, Japan), and the clinical severity of keratitis of individually scored mice was recorded by a blinded observer. The scoring system was as follows: 0, normal cornea; +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity but iris visible; +4, opaque cornea and corneal ulcer; +5, corneal rupture and necrotizing stromal keratitis. The severity of angiogenesis was recorded as described previously [20
]. In reference to the angiogenic scoring system, the method relied on quantifying the degree of neovessel formation based on three primary parameters: 1) the circumferential extent of neovessels (as the angiogenic response is not uniformly circumferential in all cases); 2) the centripetal growth of the longest vessels in each quadrant of the circle; and 3) the longest neovessel in each quadrant was identified and graded between 0 (no neovessel) and 4 (neovessel in the corneal center) in increments of 
0.4 mm (radius of the cornea is 1.5 mm). According to this system, a grade of 4 for a given quadrant of the circle represents a centripetal growth of 1.5 mm toward the corneal center. The score of the four quadrants of the eye were then summed to derive the neovessel index (range, 0 to 16) for each eye at a given time point.
Flow cytometric analysis
Single-cell suspensions were prepared from the SK- or CpG-implanted corneas. Briefly, two corneas per group were dissected, pooled together, and digested with 60 U/ml Liberase (Roche Diagnostics, Alamenda, CA) for 60 min at 37°C in a humidified atmosphere of 5% CO2. After incubation, the corneas were disrupted by grinding with a syringe plunger on a mesh, and a single-cell suspension was made in complete RPMI 1640 medium. Next, the single-cell suspension obtained from corneal samples was stained for FACS. Briefly, single cells were first blocked with an unconjugated anti-CD32/CD16 mAb (BD Biosciences, San Jose, CA) for 15 min at 4°C in FACS buffer and stained by anti-CCR2-PE, CXCR2-PE (R&D Systems), F4/80-FITC, Gr-1-FITC, or CD11b-FITC antibody (BD Biosciences) for 30 min. Detection of CD68 required fixation in PBS-buffered formaldehyde and permeabilization before staining with rat anti-mouse CD68-PE antibody (Serotec, Raleigh, NC). Finally, the cells were washed three times, and samples were acquired on a FACScan (BD Biosciences). The data were analyzed using the CellQuest 3.1 software (BD Biosciences).
Intrastromal corneal injection and micropocket assay
A nick in the epithelium and anterior stroma of a mouse cornea was made in the midperiphery with a 32-gauge needle (Becton Dickinson, Franklin Lakes, NJ) under direct microscopic observation. A blunt 32-gauge needle with a 30° bevel was introduced into the corneal stroma and advanced to the corneal center. 2 µl (0, 100, 200 ng) of recombinant MCP-1 or VEGF protein (R&D Systems) was injected under pressure into the stroma to separate corneal lamellae and disperse the protein.
In vivo angiogenic activity was assayed in the avascular cornea of mouse eyes, as described previously [9
]. Briefly, pellets for insertion into the cornea were made by combining known amounts of CpG ODNs, sucralfate (10 mg, Bulch Meditec) and hydron polymer in ethanol (120 mg/1 ml ethanol, Interferon Sciences, Elmsford, NY), and applying the mixture to a 15 x 15 mm2 piece of synthetic mesh (Tekto, Munich, Germany). The mixture was allowed to air dry, and fibers of the mesh were pulled apart, yielding pellets containing 1 µg of CpG ODNs. Pellets containing CpG ODNs were implanted into an intracorneal pocket (1 mm from the limbus). The eyes were then evaluated for corneal neovascularization. The extent of the neovessel ingrowth was recorded by direct measurement using calipers (Symbol of Quality, biomedical research instruments, Rockville, MD) under stereomicroscopy (Leica Microsytems, Wetzlar, Germany). The length of the neovessels originating from the limbal vessel ring toward the center of the cornea and the width of the neovessels presented in clock hours were measured. Each clock hour is equal to 30° at the circumference. The angiogenic area was calculated according to the formula for an ellipse. A = [(clock hours) x 0.4 x (vessel length in mm) x 
] / 2.
Cytokine assay and lymphoproliferation assay
For cytokine (IFN-
, IL-4, IL-10) assay, splenocytes and draining lymph node cells (DLNs) from mice were suspended in 10% RPMI-1640, and 106 cells in 1 ml were stimulated in vitro with irradiated syngeneic-enriched DC pulsed with UV-inactivated HSV (MOI, 1.5 before UV inactivation). A similar number of cells was Con A-stimulated (5 µg/106 cells/ml) in 96-well plates. Plates were incubated at 37°C for 72 h. The supernatant fluid was collected and stored at –80°C until use. These supernatants were screened for the presence of IFN-, IL-4, and IL-10 by ELISA. For lymphoproliferation assay, 18 h before harvesting, [3H]thymidine was added to the cultures, and cpm was measured.
Statistical analysis
Significant differences between groups were evaluated by using the Student’s t test. P < 0.05 was regarded as a significant difference between two groups.
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Figure 1. MCP-1 expression in the HSV-infected cornea. WT mice were infected with 105 PFU of HSV on their scarified corneas. At different time points, mRNA was extracted and reverse transcribed into cDNA. The labeled cDNA probes were hybridized to the cDNA GEArray membranes (A, day 1 postinfection). Arrows indicate position of MCP-1 that was upregulated in array. Kinetic expression of MCP-1 mRNA (B) and protein (C) was measured by RT-PCR and ELISA, respectively. Immunohistochemical staining of MCP-1 protein was performed, and arrows indicate the MCP-1-expressing cells in the stromal area of the SK cornea (D).
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Figure 2. MCP-1–/– mice have higher SK lesion severity, incidence, and ocular angiogenesis. Corneas of MCP-1–/– and WT mice were infected with 1 x 105 doses of HSV, and the levels of SK severity (A) and neovascularization (B) were measured in the corneas by biomicroscopy in groups of MCP-1–/– (•) and WT (o) mice. The data are compiled from three independent experiments. Statistically significant differences in SK and angiogenic score were observed between the groups (*, P<0.05; **, P<0.01). Images (C) were taken by stereomicroscopic imaging system at days 3 and 10 after virus infection (original magnification, x40). The incidence (D) of clinical SK lesions was considered as over score 2 at day 14 postinfection.
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Figure 3. MCP-1 expression in SK cornea does not influence recruitment of Gr-1+ or CD11b+ inflammatory cells expressing CCR2 and does not induce angiogenesis. Eyes of MCP-1–/– and WT mice were infected with 1 x 105 doses of HSV. Corneal samples were collected on day 2 postinfection and stained with anti-mouse Gr-1+ or CD11b+-FITC Ab in combination with a PE-antibody against CCR2 (A). Values shown in each plot reflect the percentage of inflammatory cells (Gr-1+ or CD11b+) expressing MCP-1 receptor, CCR2. Dot plots shown are representative of three similar experiments. Furthermore, corneal samples were collected on day 2 and 7 postinfection and stained with anti-mouse F4/80-FITC Ab in combination with a PE-antibody against CCR2 or CXCR2 (B). Values shown in each plot reflect the percentage of inflammatory cells expressing F4/80. Eyes of WT mice were injected with different concentrations (0. 100, 200 ng/2 µl/eye) of recombinant MCP-1 or VEGF protein, as described in Materials and Methods. The angiogenic area (C) was measured at days 2 and 4 after the MCP-1 protein injection.
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Increased recruitment of inflammatory cells expressing Gr-1 or/and CD11b to the SK cornea of MCP-1–/– mice
It was initially reasoned that the increased SK lesions of MCP-1–/– mice could result from high viral loads in SK corneas since MCP-1 deficiency might lead to decreased numbers of inflammatory cells involved in viral clearance. However, no significant differences in viral titers in MCP-1–/– mice were noted compared with WT mice (data not shown). In contrast to our hypothesis, MCP-1–/– mice showed an increased percentage of Gr-1+ or CD11b+ inflammatory cells in the SK corneas at days 1–8 after virus infection (Fig. 4A
). MCP-1–/– mice had an increased proportion of Gr-1-high cells (granulocytes) subpopulation present in the SK cornea compared with WT mice. Interestingly, an increased percentage of CD11b+/Gr-1+ cells, presumably myeloid precursor cells [25
], were observed in the SK corneas of MCP-1–/– mice at day 8 postinfection (Fig. 4B)
. In addition, proportion of CD11b+ or Gr-1+ cells expressing CD68, the monocyte/macrophage/dendritic cell lineage marker, was similar between the groups, but percentage of Gr-1+/CD68– (neutrophils) and CD11b+/CD68– (myeloid lineage) was increased in the SK cornea of MCP-1–/– mice at day 1 postinfection (Fig. 4C)
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Figure 4. The absence of MCP-1 in SK cornea enhances the recruitment of inflammatory cells. Infiltration of Gr-1+ or CD11b+ inflammatory cells in SK corneas of MCP-1–/– and WT mice was examined at different time points after virus ocular infection. Corneas from both groups were collected on each day post-HSV infection and stained with anti-mouse Gr-1+ or CD11b+-FITC Ab. Histograms are representative of three similar experiments, and values reflect the percentage of inflammatory cells expressing the respective marker (A). Infiltration of CD11b+/Gr-1+ cells in SK corneas of both groups was examined at day 8 postinfection (B). Infiltration of inflammatory cells expressing CD68 was examined. Corneas from both groups were collected on day 1 postinfection and stained with anti-mouse Gr-1+ or CD11b+-FITC Ab and anti-mouse CD68-PE Ab (C).
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, the potent chemoattractants for recruitment of inflammatory cells [18
]. As is evident in Fig. 5
, the levels of MIP-2 protein were significantly higher in SK corneal extracts from MCP-1–/– compared with WT mice at days 2 and 4 post infection (p < 0.01). Furthermore, the levels of MIP-1 protein were generally higher in SK cornea of MCP-1–/– compared with WT mice, but the difference was only significant at day 4 post infection. This indicates that higher levels of MIP-2 could explain the increased recruitment of inflammatory cells into the cornea and subsequently more severe SK in MCP-1–/– mice.
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Figure 5. Increased levels of MIP-2 and MIP-1 in the SK cornea of MCP-1–/– mice. At various time points, four corneas/group that were infected with 1x105 PFU HSV were processed to measure the MIP-2 and MIP-1 protein levels. Levels of each protein were estimated from supernatants of corneal lysates of mice by an antibody capture ELISA, as outlined in Materials and Methods. Results are expressed as means ± SE of three individual experiments. Statistically significant differences in each protein levels (*, P<0.05; **, P<0.01) were observed between the groups.
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Figure 6. Increased recruitment of inflammatory cells expressing CXCR2 in the SK cornea of MCP-1–/– mice. Expression of CXCR2 on inflammatory cells in SK corneas of MCP-1–/– and WT mice was examined at day 2 after virus ocular infection. Corneas from both groups were collected and stained with anti-mouse Gr-1+ or CD11b+ FITC Ab in combination with a PE-Ab against CXCR2. Values shown in each plot reflect the percentage of inflammatory cells (Gr-1+ or CD11b+) expressing MIP-2 receptor, CXCR2. Dot plots shown are representative of three similar experiments.
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Figure 7. Increased levels of inflammatory molecules in the SK cornea of MCP-1–/– mice. At various time points, 4 corneas/group that were infected with 1x105 PFU HSV were processed to measure the VEGF and IL-1β protein levels. Levels of each protein were estimated from supernatants of corneal lysates of mice by an antibody capture ELISA, as outlined in Materials and Methods. Results are expressed as means ± SE of four individual experiments. Statistically significant differences in each protein level (*, P<0.05; **, P<0.01) were observed between the groups.
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, or IL-1β protein were measured in the MCP-1–/– or WT corneas at days 3 and 6 after CpG implantation. Similar to the results of SK model, the levels of several inflammatory mediators were higher in the MCP-1–/– compared with WT corneas at day 3 after CpG implantation (Fig. 8B
, P<0.05). To determine whether increased production of inflammatory molecules was caused by increased infiltration of inflammatory cells, corneal cells from the CpG-implanted eye were stained with antibody against Gr-1 and CD11b. As evident in Fig. 9A
, higher numbers of Gr-1+ or CD11b+, as well as CXCR2-positive (not depicted) inflammatory cells were measured in the CpG-stimulated corneas of MCP-1–/– mice. Furthermore, increased infiltration of CD11b+/Gr-1high myeloid precursor cells, as well as CD11b+/Gr-1mid cells was observed in the CpG-implanted corneas of MCP-1–/– mice (Fig. 9B)
. The above observations indicate that the absence of MCP-1 induces more severe corneal inflammation by overexpression of other chemokines and proinflammatory cytokines.
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Figure 8. Increased CpG ODN-induced inflammation via enhanced expression of inflammatory molecules. Corneas of MCP-1–/– mice or WT mice were implanted with 1 µg of CpG pellets. The angiogenic area (A) was measured at days 3 and 6 after the CpG pellet implantation. Images were taken by stereomicroscopic imaging system at day 6 after CpG pellets implantation (original magnification, x40). At days 3 and 6 after CpG pellet implantation, 4 corneas/group were processed to measure the VEGF, MIP-2, IL-1β, and MIP-1 protein levels (B). Levels of each protein were estimated from supernatants of corneal lysates of mice implanted with CpG ODN (1 µg) by an antibody capture ELISA, as described in Materials and Methods. Results are expressed as the means ± SE of three individual experiments (4 corneas per group). Statistically significant differences in angiogenic areas (A), and VEGF protein levels (B) (*, P<0.05; **, P<0.01) were observed between the groups.
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Figure 9. Increased recruitment of inflammatory cells in CpG ODN-implanted cornea of MCP-1–/– Gr-1+ or CD11b+ inflammatory cells in CpG-stimulated corneas of MCP-1–/–, and WT mice were examined at days 3 and 6 after pellet implantation. Corneas from both groups were collected on each day postimplantation and stained with anti-mouse Gr-1+ or CD11b+-FITC Ab (A), or both (B). Data shown are representative of three similar experiments, and values reflect the percentage of inflammatory cells expressing the respective marker.
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) and Th2 (IL-4 and IL-10) CD4+ T cell responses to HSV infection in MCP-1–/– and WT mice, no significant differences could be demonstrated (data not shown). Furthermore, similar levels of HSV antigen-specific lymphoproliferation were measured in both groups (data not shown). |
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A similar study regarding MCP-1-CCR2-dependent inflammatory cell recruitment in a corneal cauterization model has also been reported [24 ]. Depletion of CCR2 or MCP-1 reduced the recruitment of F4/80-positive macrophages, but prominent infiltrates of neutrophils were also observed in both knockout and WT mice. A similar observation was also made in the current SK study. One interesting finding in our study was that more severe inflammation occurred in the absence of MCP-1, likely because of overexpression of MIP-2 and other inflammatory mediators. Because only minor populations of F4/80-positive macrophages or Gr-1+/CD11b+ inflammatory cells expressed CXCR2 or CCR2, respectively, recruitment of each cell type may not be influenced by MIP-2 or MCP-1, respectively.
Previous studies have suggested that MCP-1 plays a pivotal role in the immune modulation effects, inducing Th-2-type immune response under several pathological conditions [13 , 16 , 28 , 29 ]. Since herpetic SK is caused by Th-1-type CD4+ T cells and its severity is modulated by Th-2-type cytokines [30 , 31 ], a change in the pattern of cytokine production in response to HSV ocular infection in MCP-1–/– and WT mice was a possible explanation for the changed phenotype. This hypothesis could not be supported because both MCP-1–/– and WT mice developed similar pattern of Th1- and Th2-type immune responses. In addition to the modulatory role of MCP-1 in adaptive immune response, MCP-1 is known to recruit innate immune cells expressing CCR2 into inflammatory lesions [11 , 32 ]. Furthermore, MCP-1 induces angiogenesis caused by the production of angiogenic factors from recruited inflammatory cells expressing CCR2 [22 , 33 , 34 ]. These studies indicate that MCP-1 is strongly chemoattractant for inflammatory cells in a CCR2-dependent manner. Initially, we expected that MCP-1 upregulation in the SK cornea might be responsible for the recruitment of inflammatory cells, but most HSV-induced ocular inflammatory cells were shown to lack the CCR2 in both WT and MCP-1–/– animals. Moreover, intrastromal injection with different concentrations of recombinant MCP-1 protein into naïve cornea did not induce angiogenesis. These data made it unlikely that the recruitment of inflammatory cells was dependent on chemoattractive function of MCP-1 in the SK cornea.
Recently, a cDNA microarray comparison of expression pattern of various chemokines and cytokines was performed in inflammation model using MCP-1–/– and WT mice [17
]. Similar to our current findings, depletion of MCP-1 led to multiple perturbations of the chemokine network in vitro and in vivo, up-regulating RANTES, MIP-1, β, and MIP-2. Among these molecules, MIP-2 is a potent chemoattractant for neutrophils expressing CXCR2 in several inflammation models, including herpetic SK [6
]. Therefore, the loss of MCP-1 might be compensated by MIP-2 or other chemoattractants in the inflamed cornea. However, it is not clear whether this compensatory mechanism is performed within a single cell type or involves a complex interaction between different cell types in the cornea. Thus it was reported that MCP-1 is produced by multiple cell types, including fibroblasts, endothelial cells, muscle cells, keratinocytes, macrophages, and neutrophils [12
, 35
, 36
]. Furthermore, it would be interesting to investigate whether exogenous treatment of MCP-1 can negatively regulate multiple chemokines or if blocking MCP-1 signaling with neutralizing antibody can induce overexpression of other chemokines. The precise mechanism by which MCP-1 depletion exerts compensatory mechanism remains unresolved, and this issue is under further investigation.
In summary, our studies indicate that the successful recruitment of inflammatory cells via CXCR2 and MIP-2 is closely associated with the absence of MCP-1 in the SK cornea. MCP-1–/– mice displayed increased recruitment of inflammatory cells expressing Gr-1 or/and CD11b into the cornea and resulted in more severe angiogenesis and SK lesions. These results indicate that single-gene depletion might influence the complex chemokine and cytokine networks and further affect pathogenesis of inflammatory diseases, including herpetic SK.
Received April 30, 2006; revised August 29, 2006; accepted September 1, 2006.
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