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Macrophage Depletion Diminishes Lesion Size and Severity in Experimental Choroidal Neovascularization

¹From the Bascom Palmer Eye Institute, Department of Ophthalmology, The University of Miami School of Medicine, Miami, Florida; and the ²National Eye Institute, National Institutes of Health, Bethesda, Maryland.

	Abstract

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PURPOSE. Macrophage recruitment to the choroid has been proposed to contribute to the pathogenesis of choroidal neovascularization (CNV) in AMD. The study was conducted to determine whether treatment with clodronate liposomes (CL₂MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV.

METHODS. Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL₂MDP-lip intraperitoneally and subcutaneously 72 and 24 hours before and every 2 to 3 days after laser injury. Control mice received injections of either PBS alone or PBS liposomes. Blood monocyte and choroidal macrophage depletion were documented by flow cytometry and choroidal flatmount preparation analysis, respectively. Two weeks after laser injury, mice were injected intravenously with fluoresceinated dextran. The right eyes were removed and prepared for flatmount analysis of CNV surface area (in relative disc areas or DA), vascularity (relative fluorescence), and cellularity (propidium iodide stain). The mice were then perfused with 10% formaldehyde, and the left eyes were removed for histopathology. The means of the various parameters for four CNV lesions per eye were calculated. Fluorescein angiography was also performed.

RESULTS. Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL₂MDP-lip–treated mice. Compared with the control, flatmount analysis of macrophage depleted mice demonstrated a significant reduction in size of the CNV area (2.8 ± 0.5 DA vs. 1.4 ± 0.1 DA; P < 0.043). The treated group also revealed less vascularity (1.6 ± 0.1 units vs. 1.1 ± 0.0 units; P < 0.0092) and cellularity of CNV lesions (3.3 ± 0.6 DA vs. 1.7 ± 0.1 DA, P < 0.04). Histopathology revealed that, in the macrophage-depleted group, CNV was smaller in diameter (1270 ± 73 pixels vs. 770 ± 82 pixels, P < 0.0006) and thickness (120 ± 7 pixels vs. 96 ± 7 pixels, P < 0.019).

CONCLUSIONS. Macrophage depletion using CL₂MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.

Selective depletion of macrophages in vivo can be achieved with dichloromethylene diphosphonate-liposomes (CL₂MDP-lip).¹³ The liposomes are ingested by the macrophages, which are then destroyed after phospholipase-mediated disruption of the liposome and intracellular release of CL₂MDP. The exact mechanism of macrophage depletion by intracellular accumulation of CL₂MDP-lip is unknown, but it is believed that the intralysosomal accumulation of CL₂MDP generates signals to induce macrophage apoptosis.¹⁴ The purpose of this study was to determine whether macrophage depletion by CL₂MDP diminishes the severity of experimental CNV. Our results indicate that macrophage depletion in aged-mice decreased the severity of CNV, with the decrease defined as smaller vascular and cellular surface area and less vascularity, in an experimental model of laser CNV.

	Materials and Methods

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Mice
Mice used in this study were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-four female, normal C57BL/6 mice aged 16 months (n = 22, weight, 25–30 g), at the onset of the study, were purchased from the National Institute on Aging (Bethesda, MD). Also, seven BALB/c retired breeders were used for the immunostaining protocol, because uveal tract wholemount preparations are more informative when applied to albino animals in which uveal tissues are sufficiently thin and transparent to allow transillumination and resolution of individual stained cells.

Preparation of CL₂MDP-lip
CL₂MDP-lip was prepared ¹³ by dissolving 1,2 dioleolyl-sn-glycero-3-phosphocholine and cholesterol (both from Avanti Polar Lipids, Alabaster, AL) in a mixture of methanol and chloroform (1:1). After low-vacuum rotary evaporation at 37°C to remove the organic phase, the lipids were mixed with clodronic acid (1.9 g) dissolved in phosphate-buffered saline (0.6 M). They were allowed to sit at room temperature for 2 hours in a nitrogen-purged chamber to induce liposome swelling. Resuspension of liposomes was achieved by water sonication at room temperature for 3 minutes. The resultant liposomes were washed at 10,000g to 100,000g in an ultracentrifuge twice for 30 minutes at 16°C. The milky liposomes were removed gently with a pipette, resuspended in 4 mL of sterilized PBS, and stored in a nitrogen chamber for up to 1 week. For the PBS-liposomes the lipids were mixed with sterilized PBS.

Macrophage Depletion
Mice were anesthetized with an intramuscular administration of ketamine hydrochloride (42.8 mg/kg), xylazine (8.5 mg/kg), and acepromazine (1.4 mg/kg). Splenic and systemic macrophage depletion (C57BL/6 mice, n = 8) was performed with CL₂MDP-lip (200 µL = 1 mg) by intraperitoneal (IP) administration 4 days and 24 hours before the laser procedure and afterward every 2 to 3 days for 2 weeks. Macrophage depletion from draining lymph nodes located at the level of the submandibular, axillary, and inguinal regions was performed bilaterally by injecting subcutaneous (SC) CL₂MDP-lip (50 µL = 0.25 mg) 24 hours before laser application and afterward every 2 to 3 days for 2 weeks. Control groups (C57BL/6 mice, n = 8 each) received IP and SC administration of PBS alone or PBS-liposomes injections.^{15 16 17}

Choroidal Flatmount Preparation and Immunostaining Protocol
Briefly, whole-body perfusion was performed with (1x) PBS+K₃ EDTA (1.7 mg/mL) followed with fixation with 4% paraformaldehyde. Enucleation of the eyes was performed, and removal of the anterior segment and neurosensory retina was performed after fixation. The remaining eye cup was placed on a glass slide, and three to four relaxing incisions were made through the optic disc, leaving three to four pieces of choroid and sclera, respectively. The choroid was separated from the underlying sclera with a Beaver blade no. 64.¹⁸ The choroid was permeabilized and hydrated. F4/80 antibodies (10 µg/mL MCA497R; Serotec, Raleigh, NC) were used, followed by incubation with a secondary biotinylated antirat antibody (ABC Kit; Vector Laboratories, Burlingame, CA). A peroxidase substrate (Vector VIP, Vector Laboratories) was used until the reactions in the binding sites were developed to the desired intensity. Macrophage density morphometry was performed after digitizing the choroidal flatmount preparations with a light microscope (x400 magnification) connected to a color video camera and a frame grabber. Three representative sections were analyzed at high-power magnification per piece of choroidal tissue, and the results were averaged with those in the remainder of the pieces to obtain the density of macrophages per eye. This was compared between macrophage-depleted animals (BALB/c, n = 3) and the control (BALB/c, n = 4; PBS or PBS-liposomes).

Monocyte Isolation and Flow Cytometric Evaluation
Briefly, white blood cells were separated from EDTA-anticoagulated whole blood by gradient separation technique.¹⁹ For flow cytometry, the monocytes were labeled with a rat anti-mouse F4/80 antigen-FITC conjugate (10 µg/mL MCA497F; Serotec). Cells were prepared for analysis in flow cytometer using digitonin (1 mg/mL) and propidium iodide (PI, 500 µg/mL) modified from previous publications.²⁰

Laser treatment, fluorescein angiography, histology, and flatmount preparation and analysis were performed as described in an earlier published study.¹

Statistical Analysis
Morphometric data for different lesions in each eye were averaged to provide one value per eye. The mean ± SD of these measures for each group was calculated and probabilities (t-test) were obtained on computer (Prism, ver. 3.0; GraphPad, San Diego, CA). P < 0.05 was considered statistically significant in all forms of statistical analysis used.

	Results

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First, we confirmed that CL₂MDP-lip treatment adequately depleted circulating monocytes and choroidal macrophages. Flow cytometry was used to determine the degree of depletion of circulating monocytes. The total leukocyte population isolated from whole blood was stained for F4/80, a mouse macrophage-specific marker. Because monocytes constitute only 2% to 8% of total blood leukocytes, we enriched the sample for monocytes by using flow cytometry to identify the subset of large leukocytes (consisting of monocytes and large granular lymphocytes). Within this subset, leukocytes from mice treated with PBS or PBS-liposomes typically demonstrated approximately 60% positivity for F4/80. In contrast, CL₂-MDP-lip treatment reduced the percentage of F4/80-positive cells to 15%, indicating a reduction of approximately 75% (Fig. 1) .

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Flow cytometry of white blood cells obtained from mouse blood. (A) Dual light-scatter characteristics for the gated population of macrophages and large lymphocytes. Acquisition gates were set to detect only the PI fluorescence-positive events from a cell suspension in which no antibody staining was used. (B–D) Positive staining of blood monocytes in the gated population after labeling them with the macrophage marker F4/80 after treatment with PBS (B), PBS-liposomes (C), or CL2MDP-lip (D). CL2MDP-lip–treated mouse showed depletion of monocytes to 15%.

View larger version (76K): [in this window] [in a new window]   FIGURE 2. Choroidal flatmount preparations from BALB/c mouse for detection of choroidal macrophages using anti-F4/80 staining. (A) Choroidal macrophage density in a control mouse compared with the diminished density of macrophages (B) in which the animals received treatment with CL2MDP-lip for 2 weeks. Magnification, x400.

View larger version (50K): [in this window] [in a new window]   FIGURE 3. Fluorescein angiograms of eyes obtained 2 weeks after diode laser photocoagulation to induce CNV. (A–C) angiogram of a control (PBS treated) animal. (A) A pointlike hyperfluorescent area that greatly increased in size and intensity, as shown in (B) and (C). There was intense leakage in the late phases (1- and 5-minute frames, respectively corresponding to intermediate [B] and late [C] phases) of the angiogram which shows the severity of the CNV lesion. (D–F) Fluorescein angiogram of a treated (CL2MDP-lip) animal. A similar angiographic pattern can be seen but the intensity and the size of the hyperfluorescent lesion is smaller. (D) Depicts the initial phase of the angiogram in which a pointlike hyperfluorescence is evident, which grows moderately in size and intensity during intermediate (E) and late (F) phases of the angiogram.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Quantitative analysis of histopathology sections. (A) Maximum lesion diameter (measured in pixels) in which the CL2MDP-lip treated group shows significantly smaller diameter than the control group (P < 0.0006). (B) Maximum lesion thickness (measured in pixels) in which the CL2MDP-liposomes treated group shows a significantly lesser lesion thickness than the control group (P < 0.03). *Statistically significant difference.

View larger version (85K): [in this window] [in a new window]   FIGURE 5. Flatmount preparations of the posterior pole of a mouse eye using FITC-dextran and PI stains 2 weeks after diode laser photocoagulation. (A, B) Vascularity and cellularity of the lesions of the control group. CNV lesions were large and with irregular borders. (C, D) Small discrete circular lesions (dotted circles) of the CL2MDP-lip–treated animals. The size, vascularity, and cellularity of the lesions were less in the macrophage-depleted mice (Fig. 6) . D, optic disc.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Quantitative analysis of flatmount specimens, showing that CL2MDP-lip–treated animals had significantly less severe CNV lesions when measured with FITC and PI staining. (A) Vascular margins (as measured in disc areas) of the lesions in both the control and the treated mice. Compared with control animals, analysis of flatmounted retinas of macrophage depleted mice demonstrated significant reduction in size of CNV lesions (2.8 ± 0.5 DA vs. 1.4 ± 0.1 DA, P < 0.043). (B) Vascular luminosity index. The treated group also revealed less vascularity of CNV lesions (1.6 ± 0.1 units vs. 1.1 ± 0.0 units, P < 0.0092). (C) Cellular margins (size), measured in DAs in which the treated group also showed a decrease in size compared with the nontreated one (3.3 ± 0.6 DA vs. 1.7 ± 0.1 DA, P < 0.04). (D) Cell density was reported as the cell-density index, which was not significantly different between groups. Nevertheless, the total cellular component of the depleted animals was less due to the decrease in size of the CNV. *Statistically significant difference.

	Discussion

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Macrophages in tissues are derived from circulating blood monocytes. Within tissue they undergo maturation, adaptation to their local microenvironment, and differentiation into various types of cells that perform specific housekeeping, trophic, and immunologic functions. Macrophages play an important role in pathogen recognition and clearance, as well as scavenging of senescent and dying cells. They also function as antigen-presenting cells for T lymphocytes and as inflammatory effector cells. Several different stages of metabolic and functional activities, which in turn, represent different programs of gene activation are often described, including scavenging and activated macrophages.²¹ Activated macrophages are most efficient at synthesizing and releasing mediators to amplify inflammation and can mediate chronic injury leading to processes such as fibrosis, wound repair, extracellular matrix turnover, and angiogenesis.^{22 23}

In studies using rats, macrophage subpopulations are better delineated by monoclonal antibodies.²⁴ The ED-1 positive, blood-derived macrophage population represents short-lived cells with high turnover that are recent recruits to tissues. These cells are thought to serve inflammatory and scavenging functions. In contrast, the ED-2 tissue-resident subset comprises relatively long-lived cells with low turnover and long duration in tissues. These cells are thought to serve trophic and tissue-maintenance functions.²⁵ Dendritic cells are specialized noninflammatory monocytes that present antigen to naïve T cells.²⁶

The normal choroid is richly invested with various subsets of monocytes and macrophages, including blood-derived macrophages, tissue-resident macrophages and dendritic cells.^{27 28} However, monocytes and macrophages are difficult to evaluate by histology in normal or diseased choroid, because they acquire flattened profiles that cause them to be difficult to demonstrate on routine histologic sections. Nevertheless, they are presumed to serve similar functions in the choroid, as described in other tissues.

Grindle and Marshall²⁹ were probably the first to postulate a role for macrophages in the pathogenesis of AMD. Penfold et al.^{30 31} subsequently showed the involvement of low-grade chronic inflammatory cell infiltration in AMD, and Sarks et al.³² and Penfold et al.³³ reported macrophages and giant cells in association with Bruch’s membrane, geographic atrophy, and CNV. Most recently, Anderson et al.³⁴ have suggested that many large drusen appear to contain a central core derived from a choroidal monocyte, perhaps representing a dendritic cell.

Many investigators have suggested that macrophages might be a stimulus for CNV and a common factor linking the diverse diseases associated with CNV.³⁵ Grossniklaus et al.^{10 36} have demonstrated significant macrophage infiltration in specimens of surgically excised CNV and that these cells appear to be synthesizing various cytokines and growth factors. In a primate model for laser-induced experimental subretinal neovascularization, Ishibashi et al.³⁷ showed that macrophages migrate into the laser wounds and seemed to contribute to the neovascular response. Finally, ocular corticosteroid therapy has been used in the treatment of CNV. Although corticosteroids inhibit many cellular processes they are potent modulators of macrophage functions. These studies^{38 39 40} suggest that more research is needed to explore the therapeutic potential of nonspecific anti-inflammatory therapy in AMD. Specifically, triamcinolone acetate has been used to treat CNV lesions, based on research in which these drugs have the potential to influence cellular permeability and downregulate the expression of inflammatory markers.^{41 42} Taken together, these observations imply a potential pathogenic role for cytokines, chemical mediators, matrix metalloproteinases, mitogens, or angiogenic factors released by macrophages from the choroid.^{10 12}

In this study, we evaluated the effect of macrophage depletion on the severity of experimental CNV in aged (16 months) old mice. Aging has been shown to increase the severity of CNV lesions in mice.¹ We found that CNV in macrophage-depleted mice were smaller, thinner, less cellular, and less vascularized than nondepleted mice but did not decrease the incidence of CNV. However, we did not observe a difference in the cell density or frequency of actively leaking lesions between compared groups. Nevertheless, the total cellular component of the depleted animals was less because of the decrease in size of the CNV.

The effective depletion of macrophages was confirmed by flow cytometry of circulating monocytes and microscopic examination of choroidal flatmount preparations for choroidal macrophages. Different percentages of depletion were determined with the two analyses. The level of depletion of blood monocytes achieved in our study was consisted with published results (70%–95%).^{17 43} In other studies of nonocular tissues, depletion of tissue macrophages by clodronate has been more variable. The ED-1–positive, blood-derived macrophage population appears more susceptible to depletion by clodronate than do the ED-2–positive tissue-resident macrophages.^{44 45} Our findings seem to concur, because circulating monocytes were 75% depleted, but choroidal macrophages were only reduced by 46%. We presume that the residual choroidal macrophages represent mostly tissue-resident macrophages. If true, then our results suggest that the blood derived circulating monocytes are more likely to contribute to CNV pathogenesis.

PBS alone and PBS-liposome–treated animals were used as control subjects for completeness. Empty liposomes, in common with other particulate compounds, may influence macrophage biology and must be used as a specificity control when liposomes are used as a drug delivery method for specific agents. However, as stated by investigators who developed this procedure, the best control for studies in which general macrophage depletion is required (as in our study) are PBS sham injections. This control would represent normal, healthy, nonblocked, nonsuppressed, and nonstimulated macrophages.¹³

Recent data from several groups suggest that innate immune mechanisms and localized choroidal inflammation may generally contribute to the pathogenesis of AMD. In addition to macrophages, other relevant innate immune mechanisms involved in AMD pathogenesis include injurious stimuli (oxidants or infectious agents) and amplification cascades (such as complement, mediators systems, and cytokines). For example, Anderson et al.³⁴ have identified complement and immune complexes in association with nodular drusen, which are potentially at high risk for CNV formation.³⁴ Perhaps immune complexes are stimuli for the recruitment of macrophages or monocytes in human AMD.

	Footnotes

 
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2002.

Supported by National Eye Institute Grant EY/AI 13318.

Submitted for publication January 14, 2003; revised February 28, 2003; accepted March 18, 2003.

Disclosure: D.G. Espinosa-Heidmann, None; I.J. Suner, None; E.P. Hernandez, None; D. Monroy, None; K.G. Csaky, None; S.W. Cousins, None

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

Corresponding author: Scott W. Cousins, Bascom Palmer Eye Institute, William L., McKnight Vision Research Center, 1638 N.W. 10th Avenue, Miami, FL 33136; scousins{at}med.miami.edu.

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