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Published online before print May 22, 2003
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Departments of
* Ophthalmology,
Cardiovascular Medicine, and
Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;
San Francisco General Hospital, Gladstone Institution of Cardiovascular Division, University of California; and
¶ Department of Ophthalmology, Kagoshima University, Japan
Correspondence: Koh-Hei Sonoda, M.D., Ph.D., Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, Japan 812-8582. E-mail: sonodak{at}med.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, was also observed. Collectively, we conclude that ocular-infiltrating macrophages play an important role in CNV generation.
Key Words: photocoagulation • age-related macular degeneration • retinal pigment epithelium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The exact cellular and molecular mechanisms that induce CNV remain to be elucidated. In a human AMD study, some macrophages surround the macular area, thus suggesting that macrophages play a role in the formation of CNV [7 8 9 10 11 ]. Vascular endothelial growth factor (VEGF) is expressed in macrophages in some experimental animal CNV models [12 , 13 ]. Recent evidence suggests that macrophages play an important role in the formation of angiogenesis in wound healing based on findings using tumor models [14 , 15 ]. Functionally, macrophages form a heterogeneous cell population. They have the ability to influence each phase of the angiogenic processes [16 ]. Macrophages are derived from blood monocytes, which enter tissues via binding to specialized vascular endothelial cells. Locally produced stimuli cytokines, adhesion-molecule binding, or interaction with foreign infectious agents promptly activate the macrophage [15 , 17 ].
Photocoagulation (PC)-induced CNV is a well-established animal model of CNV that is useful for investigating the precise mechanisms of human diseases [18 19 20 ]. To reveal the influence of macrophage infiltration in PC-induced CNV, we studied the development of experimental CNV in CCR2-deficient mice that lack macrophages at the inflammatory sites.
In this paper, we demonstrated that ocular-infiltrating macrophages play a crucial role in the development of the mouse PC-induced CNV model and that ocular-infiltrating macrophages possess a direct angiogenic ability. Together, the data provide insight into the role of ocular-infiltrating macrophages in PC-induced angiogenesis and CNV formation in general.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of CNV
PC-induced CNV was generated by a technique described previously with some modifications [20
]. Briefly, B6 mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight), and the pupils were dilated with 1% tropicamide. Dye laser PC (wavelength, 630 nm; 0.1 s; spot size, 100 µm; power 150 mW) was performed around the disc of the retina through a slit lamp delivery system using a Nidek photocoagulator. The posterior pole of the retina was thus burned while a hand-held cover slide was used as a contact lens. Only lesions in which a subretinal bubble or focal serous detachment of the retina developed were used for the experiments.
Isolation and counting of the ocular-infiltrating macrophages
To examine the macrophages in the retina and choroid, single cells were prepared from mouse eyes according to the procedure used to isolate hepatic lymphocytes, with some modifications [22
, 23
]. To collect a sufficient number of ocular-infiltrating cells, 40–50 burns were delivered to mice eyes as pan retinal PC. After PC, the eyes were enucleated, and then the anterior segment (cornea, iris, and lens) was taken out. The posterior segment of eye including sclera, choroid, and retina was disrupted with scissors and then shaken in medium supplemented with 0.5 mg/ml Collagenase type D® (Boehringer Mannheim, Germany) at 37°C for 40 min. As the basic medium, we used RPMI 1640 (Gibco Laboratories, Grand Island, NY) with 10% fetal bovine serum (Gibco Laboratories), 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 10-5 M 2-mercaptoethanol, and 5 mg/ml HEPES buffer. The supernatants were collected, passed through a metal mesh, and washed three times, and viable cells were thus obtained. At least two eyes were needed to obtain a sufficient number of viable cells for a reliable flow cytometric analysis in the above process. A total of six eyes (three individual pools) were examined per group.
Antibodies and reagents
The following reagents were used for the flow cytometric studies. Fluorescein isothiocyanate (FITC)-conjugated anti-CD45 monoclonal antibody (mAb; clone name YW62.3) was purchased from IQProducts (Groningen, The Netherlands). FITC-conjugated anti-CD40 mAb (clone name 3/23), FITC-conjugated anti-CD80 (B7-1) mAb (clone name 16-10A1), FITC-conjugated anti-CD86 (B7-2) mAb (clone name GL1), FITC-conjugated anti-intraobserver intrasession/intraobserver intersession mAb (clone name 2G9), FITC-conjugated anti-Ly-6G (Gr-1) and Ly-6C (clone name RB6-8C5), and Cy-Chrome anti-mouse T cell receptor-ß mAb (clone name H57-597) were purchased from BD PharMingen (San Diego, CA). Biotin-conjugated anti-F4/80 mAb (clone name A3-1) and biotin-conjugated rat immunoglobulin G (IgG)-2b (clone name R2b15) were purchased from Caltag Laboratory (Burlingame, CA). Streptavidin/R-phycoerythrin (PE) was purchased from Molecular Probes (Junction City, OR).
Flow cytometry
Intraocular-infiltrating cells were adjusted to the designated concentrations, then were stained by two colors with FITC–anti-CD45 mAb and biotin-conjugated anti-F4/80 mAb, and then were counterstained by Streptavidin/R-PE. Macrophages have been previously reported to be CD45 and F4/80 surface molecules-positive. Therefore, CD45 and F4/80 mAb double-stained cells were analyzed as macrophages. Flow cytometry was performed with EPICS® XL (Beckman Coulter, Mannheim, Germany). The number of ocular-infiltrating macrophages was calculated from the percent of each population in the gate of the precounted, total number of viable cells using trypan blue dye exclusion. The gate of F4/80-positive cells was set up according to the staining pattern of isotype control (biotin-conjugated rat IgG2b).
Macrophage enrichment [magnetic cell sorter (MACS) selection]
For macrophage enrichment, the cells from B6 mice were treated with biotin-conjugated anti-F4/80 mAb before magnetic bead selection. Ab-labeled cells were treated with Streptavidin MicroBeads (Miltenyi Biotec, Auburn, CA) for 15 min and were washed twice. To harvest the macrophage-enriched cells, the cells were applied to a type MS 1-positive selection column with MiniMACS (Miltenyi Biotec). Positively selected cells were stained with CD45 mAb, and enrichment was confirmed by flow cytometry. The number of cells in the enriched populations was adjusted to the approximate number used in the control studies.
As a control macrophage, alveolar macrophages were prepared as described previously [24 ]. Briefly, alveolar macrophages were collected as bronchoalveolar lavage cells (including alveolar macrophages and infiltrating monocytes) by washing the lung with a total of 10 ml of the above-described per lung (1 ml per wash). Next, the macrophages were enriched using the MACS technique as an ocular-infiltrating macrophage preparation.
Dorsal air sac (DAS) assay
The DAS assay was performed in mice as described by Oikawa et al. [25
] with some modification [26
]. MFTM membrane filters (Millipore, Ireland) were filled with cell-enriched macrophages separated from the eye by using the MACS technique as described above. At least 18–20 eyes from B6 mice were needed to obtain a sufficient number of ocular-infiltrating macrophage enrichment for a reliable reaction (
5x105 cells).
The negative-control group was implanted with chambers containing cells that had no macrophages (macrophage-eliminated cells). The blank control group was implanted with chambers containing only an equal volume of basic medium. Each chamber was implanted into a DAS of B6 mice produced by injecting 10 ml air. Five days after implantation, the chambers were removed with dorsal skin, and then a ring without filters was placed on the same site and photographed. The angiogenic response was assessed under a dissecting microscope by determining the total length of newly formed vessels. The newly formed vessels were detected because of their tendency to demonstrate loop formation and tortuosity [27 28 29 30 ].
Retinal flat-mount preparation
In a mouse experimental CNV model, neovascuralization can be detected within 1 week [20
]. To study the effect of macrophage infiltration on the development of CNV, we evaluated the early phase of CNV on day 6 using the flat-mount technique as described previously [31
]. To evaluate the size of the CNV lesions individually, four burns were performed while leaving a space (3, 6, 9, and 12 o’clock positions around the optic disc). Mice were anesthetized and perfused with 1 ml phosphate-buffered saline (PBS) containing 50 mg/ml fluorescent-labeled dextran (25,000 average molecular weight; Sigma Chemical Co., St. Louis, MO), and the eyes were removed and fixed for 30 min in 4% paraformaldehyde. The cornea and lens were removed, and then the entire retina was carefully dissected from the eyecup. Radial cuts (average, eight) were made from the edge of the eyecup to the equator, and then the eyecup was flat-mounted in an aqua-mount with the sclera facing down and the choroid facing up. Flat mounts were examined by fluorescence microscopy, and images were digitized using a three-charge-coupled device color video camera and a frame-grabber. To measure the total area of hyperfluorescence associated with each burn corresponding to the total number of fibrovascular scars, MacScope (version 2.3; Mitani, Fukui, Japan) was used.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from enriched macrophages 48 h after PC using Trizol® (Invitrogen, Carisbad, CA). Total RNA was reverse-transcribed and amplified by the Gene Amp PCR System 9600 (Perkin-Elmer, Norwalk, CT). First-strand cDNA was synthesized using avian myloblastosis virus RT (Boehringer Mannheim, Indianapolis, IN). The incubation time was 10 min at 25°C and 60 min at 42°C. RT was denatured at 99°C for 5 min before PCR amplification. cDNA was subjected to PCR in a 20-µl volume containing 10 pmol primer pair and 2 µl light cycler (Roche Molecular Biochemicals, Indianapolis, IN). The reaction conditions were as follows: denaturing at 95°C for 60 s, followed by 40 cycles of denaturing at 95°C for 0 s, annealing at 56°C for 10 s, and extending at 72°C for 10 s. The PCR products were electrophoresed on 2% agarose gel and then were stained with ethidium bromide. The amount of RNA in each sample was standardized by preliminary amplification for ß-actin and readjusting the sample concentration according to densitometry reading of ß-actin bands, as described above. The adjusting systems were repeated until the ß-actin bands were equalized in serially diluted samples.
The primers used in these experiments are listed below. For amplification: ß-actin, sense 5'-GTG GGC CGC TCT AGG CAC CAA-3' and antisense 5'-CTC TTT GAT GTC ACG CAC GAT TTC-3' (product size: 539 bp); basic fibroblast growth factor (bFGF), sense 5'-ACA GGT CAA ACT ACA ACT CCA -3' and antisense 5'-TCA GCT CTT AGC AGA CAT TGG -3' (product size: 295); VEGF, sense 5'-TTA CTG CTG TAC CTC CAC C-3' and antisense 5'-ACA GGA CGG CTT GAA GAT G -3' (product size: 189 bp); tumor necrosis factor (TNF-), sense 5'-GGC AGG TCT ACT TTG GAG TCA TTG-3' and antisense 5'-ACA TTC GAG GCT CCA GTG AAT TCG G-3' (product size: 309 bp). At least 10 eyes were needed to obtain a sufficient number of enriched macrophages for a RT-PCR analysis in the above process.
Histological examination
The tissue specimens were fixed in 4% paraformaldehyde and then embedded in paraffin. Next, 3-µm sections were cut perpendicularly to the retinal lesion exhibiting the thickest laser-induced retinal destruction. For immunohistochemical studies, all incubation steps were performed in a moist chamber, and all rinsing was performed by immersing the slides in a PBS bath. The sections were thereafter rehydrated with a graded series of alcohol and rinsed with PBS. Hydrogen peroxide–methanol (0.3%) was applied to each specimen for 10 min to block any endogenous peroxide activity. After incubation by blocking with 2% skim milk for 30 min, the slides were incubated overnight at 4°C with primary antibodies. For the detection of macrophages, rat anti-mouse F4/80 Ab (1:800) was used. The sections were washed again with PBS for 10 min and then were incubated with FITC-labeled secondary Ab (1:200) for 30 min. The sections were rinsed in PBS for 10 min and observed with a fluorescence microscope. As negative-control slides, for the detection of macrophages, rat nonimmunized IgG were used.
Statistics
The areas of CNVs and the total length of newly formed vessels were analyzed to identify any for significant differences among the experimental groups by Student’s t-test. A value of P ≤ 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Angiogenic ability of ocular-infiltrating macrophages
To examine the direct angiogenic activity of ocular-infiltrating macrophages in a CNV model, ocular-infiltrating cells separated from PC-treated eyes were divided into a macrophage-enriched population and a macrophage-eliminated population using magnetic beads. The quality of the separation technique was monitored by flow cytometry (Fig. 3A
). We used an in vivo mouse DAS assay to evaluate the direct angiogenic activity. The macrophage-enriched cells showed significant angiogenic responses, which were characterized by newly formed vessels, in contrast to the limited angiogenic responses induced by the macrophage-depleted cells (Fig. 3B
and 3C) . Alveolar macrophages have a special phenotype that suppresses various immune responses, expresses a low level of class II molecules, and contributes to inhalation-induced, systemic tolerance [37
]. As expected, alveolar macrophages (negative-control) also showed a limited angiogenic response (Fig. 3C)
. Furthermore, mice with implantation of chambers containing only medium (negative-control group) expressed a minimum degree of angiogenesis, supporting the conclusion that the experimental manipulation did not induce a significant angiogenic response. Thus, ocular-infiltrating macrophages induced angiogenic activity in vivo.
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. Ocular-infiltrating macrophages (eye) derived from B6 mice expressed VEGF, bFGF, and TNF-. In contrast, alveolar macrophages (lung) did not express those cytokines but instead, only expressed TNF- mRNA (Fig. 5
). These data support the conclusion that ocular-infiltrating macrophages promote the development of CNV formation by inducing angiogenic factors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CCR2 KO mice showed markedly reduced macrophage recruitment to the PC-injured site and failed to form CNV. Despite a normal number of circulating leukocytes and resident macrophages, CCR2 KO mice failed to recruit macrophages to the inflammatory site [35 , 36 ]. We carefully confirmed that the ocular infiltration of macrophages was significantly impaired in CCR2 KO mice using flow cytometry (Fig. 2) . Although there was a little discrepancy of the population of ocular-infiltrating macrophage between B6 (Fig. 1A) and CCR2 control mice (Fig. 2A) , this was a result of a difference of genetic background in control mice (hybrid of B6 and 129/Sv). This in vivo evidence strongly suggested that macrophages play a critical role in CNV formation.
Another interpretation of our data using CCR2 KO mice is also possible. As the CCR2 KO mouse is not equal to the macrophage KO mouse, the other inflammatory cells other than macrophages might also be affected by the CCR2 deficiency and contribute to CNV reduction in CCR2 KO mice. In fact, CCR2 is expressed on T cells, activated natural killer (NK) cells, and NKT cells [38 39 40 ]. However, the numbers of ocular-infiltrating cells that express T cell receptor or NK marker were usually small until 6 days after PC injury (<1.5%) in B6 mice. Additionally, in contrast to the lung virus infection model [41 ], no delayed T cell migration was observed in CCR2 KO mice in our model. The other important cells were neutrophils. Our preliminary data showed that the ratio of neutrophils in the ocular-infiltrating cells was temporally increased at 24 h after PC in CCR2 KO mice compared with control (hybrid of B6 and 129/Sv) mice; however, it came to the equal level at 48 h after PC. Although the neutrophils were reported to have the angiogenic ability [42 ], the neutrophils could not compensate the macrophage function, and CNV area was markedly reduced in CCR2 KO mice. Collectively, although we understood our CCR2 KO data were still indirect, we concluded that the contribution of the cells other than macrophages was relatively small, and the CNV inhibition in CCR2 KO mice was mainly a result of their lack of macrophage recruitment into the eye.
Although macrophages are crutial for CNV, our data do not completely rule out the possibility that other types of ocular-infiltrating cells also participate in CNV generation. Neutrophils are known to produce angiogenic factors in several models [42 ], and lymphocytes can contribute to CNV formation with the aid of activated macrophages. T lymphocytes are activated through class II and B7 molecules and produce a series of inflammatory cytokines and chemokines [43 ]. Signaling via CD40 mediates Ig–isotype switching in B lymphocytes. Melter et al. [44 ] demonstrated that the interaction between T cells and macrophages through CD40L–CD40 is potent for VEGF expression and VEGF-induced angiogenesis. Choroidal-infiltrating cells formed a network for local inflammation and thereby induced CNV.
Until now, the relationship between macrophages and angiogenesis has been mainly discussed using a malignant tumor model [14 ]. The release of angiogenic factors by tumor-associated macrophages (TAM) has been shown to play an important role in the formation of neovessels and tumor growth [45 46 47 ]. TAM are derived from blood monocytes, which enter tissues by adhering to the vascular endothelium via the interaction of ligands such as lymphocyte function-associated molecule-1 on monocytes and intercellular adhesion molecule-1 on endothelial cells [48 ]. After extravasation, monocytes undergo differentiation into macrophages and remain in tissues. TAM secrete an array of angiogenic cytokines and growth factors [49 ] in addition to proteolytic enzymes [50 ]. The accumulation of macrophages also correlates with the number of newly formed vessels after retinal vascular occlusion in human [51 ].
In this report, we demonstrated that ocular-infiltrating macrophages are a potential source of various angiogenic factors such as VEGF in the development of CNV. Our findings are compatible with other recent CNV studies using an animal model [12
, 13
]. In contrast, Oh et al. [11
] reported the pattern of soluble factors derived from infiltrating macrophages based on immunohistochemical analyses slides, surgically excised choroidal neovascular membranes, and they demonstrated that macrophages were located in the choroidal neovascular membranes produced by interleukin-1ß and TNF- but not by VEGF. The reason for this discrepancy may be a result of differences between humans and mice or of the timing of the analyses. In our studies, macrophages were isolated from mice at 48 h after PC. They were activated and possessed an angiogenic capacity by themselves. In contrast, macrophages located in the choroidal neovascular membranes needed a long time to undergo infiltration and could be substantially modified from their original phenotypes.
Recently, some reports on human CNV specimens have suggested that the CNV is a result of a nonspecific, inflammatory response [52 ]. The deposits in and around Bruch’s membrane have also been reported to possibly induce an inflammatory response, and occasionally, macrophages were identified in pathologic specimens in human AMD [7 8 9 , 53 ]. In addition, inflammation was implicated in the development of CNV in ocular histoplasmosis and multifocal choroiditis [54 , 55 ]. We not only confirmed this concept but also provide data to show that the infiltrating macrophages directly contribute to CNV formation. This new insight into the role of macrophages in CNV formation may thus lead to the development of novel, therapeutic approaches in the future, which will enable us to modify the macrophage function in human disease.
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ACKNOWLEDGEMENTS |
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Received September 6, 2002; revised January 10, 2003; accepted March 5, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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