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1From the Departments of Ophthalmology, 3Cell and Developmental Biology, 4Medicine, and 5Molecular Microbiology and Immunology, Casey Eye Institute, Oregon Health & Science University, Portland, Oregon. 2Present affiliation: Universitäts-Augenklinik, Heidelberg, Germany.
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METHODS. To label potential antigen-presenting cells, fluorescently tagged ovalbumin was injected into the anterior chambers of mouse eyes. Fluorescently tagged antibodies to cell surface proteins were injected to label specific cell types. Intravital fluorescence microscopy with digital image recording was used to visualize the labeled cells in the irises at various times after the injection.
RESULTS. The pattern and density (116–148 cells/mm2) of cells labeled in vivo by fluorescent ovalbumin or F4/80 antibodies were similar to that identified by conventional wholemount immunostaining for macrophages and dendritic cells. Fluorescent antibodies specific for CD11b, CD11c, CD80, CD86, or major histocompatibility complex (MHC) class II protein each labeled selective populations of cells in vivo. In contrast to conventional histology, in vivo immunohistology permitted serial observations. The phenotype of cells labeled by fluorescent ovalbumin was not the same at 6 (95% CD11c+) and 24 hours (24% CD11c+) after injection.
CONCLUSIONS. This method of in vivo immunohistology provides a tool for studying cell kinetics and dynamic interactions that cannot be assessed by conventional immunohistology. Furthermore, it avoids potential artifacts from tissue fixation and may work with antibodies that label cells poorly in vitro.
, are competent antigen-presenting cells.1 In 1973, Steinman and Cohn2 first described dendritic cells as highly efficient antigen-presenting cells. During the past decade, the dendritic cell has been credited as being the principle antigen-presenting cell in the activation of naïve T cells.3 Dendritic cells can carry antigen from peripheral tissues thorough lymphatic vessels to activate naïve lymphocytes in secondary lymphatic tissues (e.g., lymph nodes). Cytokine secretion by the dendritic cell is instrumental in directing the T lymphocyte to a T-helper cell type 1 (Th1) or Th2 response.4 5 6 Dendritic cells are a heterogeneous group of cells that includes members of different lineages and states of maturation.4 5 6 Some dendritic cells, as well as macrophages, are derived from myeloid progenitor cells. Other dendritic cells develop from a lymphoid lineage. Dendritic cells have an immature phenotype when they enter the peripheral nonlymphoid tissues and are characterized as highly phagocytic cells with abundant MHC class II proteins in intracellular compartments. In response to a maturation signal, the cells mobilize the MHC class II proteins to the cell surface, greatly increase expression of lymphocyte costimulatory proteins, become less phagocytic, and change their chemokine receptor profile to guide them to lymphatic vessels and then nodes, wherein they can prime naïve lymphocytes.
Most of the characterization of macrophage and dendritic cell subtypes has been in vitro. The cell’s lineage, state of maturity, and microenvironment all affect the cell’s phenotype, as evidenced by variability in the expression profiles of cell surface proteins and cytokines. Because different dendritic cell subtypes can coexist in the same tissue and have overlapping properties, determining their precise roles in an immune response within that tissue requires experiments that have the power to distinguish each cell type in vivo. This resolution would help determine, for example, whether macrophages, dendritic cell subsets, or both present antigens to T cells recruited to sites of inflammation.
Immunohistology is a widely used technique to identify cell phenotypes in vitro. In this report, we describe the use of fluorescently labeled antibodies to cell surface proteins and fluorescently labeled antigen for immunohistology studies in vivo. These studies are a modification of the intravital fluorescence microscopy technique that we have used to study leukocyte migration in and around blood vessels in different tissues of the eye.7 8 9 10 Herein, we show images of living mouse iris with dendriform cells that have ingested ovalbumin and selective labeling of some of these cells by monoclonal antibodies to MHC class II, F4/80 (found on both macrophages and dendritic cells11 ), CD11b (found on macrophages and myeloid, not lymphoid, dendritic cells12 ), and CD11c (found on most murine dendritic cells13 ) antigens, and the costimulatory molecules CD80 and CD86.
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Fluorescent Labeling of Proteins
Purified protein antigens and carrier-free antibodies were labeled with fluorescent succinimidyl esters (Alexa Fluor green 488, red 594, or orange 546; Molecular Probes, Eugene, OR). The green fluorescent conjugates are significantly brighter than fluorescein conjugates and are much more photostable. The conjugations were accomplished by a simple incubation of the reagent with the protein according to the manufacturer’s instructions, followed by buffer exchange and protein concentration with ultrafiltration devices (Centricon-10; Millipore, Bedford, MA). Optical density measurements were used to calculate the efficiency of protein labeling and recovery according to the following formulas: protein concentration (M) = [A280 – (Af x Cf) x dilution factor]/
p; and moles dye per mole protein = Af x dilution factor/(f x protein concentration) (A, absorbance; , molar extinction coefficient; Cf = A280/Af for the dye). p is 203,000 for IgG and 31,000 for ovalbumin. For the green fluorescence conjugate, Af was measured at 494 nm, Cf = 0.11, and f = 71,000. For the red conjugate, Af was at 590 nm, Cf = 0.56, and f = 73,000. Optimum sensitivity with minimal background labeling was obtained when the bound dye/protein molar ratios were kept within the recommended values of 4 to 9 for the green conjugate and 2 to 6 for the red. Antibodies to MHC class II (anti I-Ad; Pharmingen, San Diego, CA), F4/80 (Serotec, Raleigh, NC), CD11b (Serotec), CD11c (Serotec), CD80 (Pharmingen), and CD86 (Pharmingen) as well as mouse (Pharmingen), hamster (Serotec), and rat (Serotec) IgG2b isotype controls were used in the study.
Anterior Chamber Injections
Anterior chamber injections of the mouse eye were performed with a Hamilton syringe to deliver a volume of 2 or 3 µL during direct visualization under a surgical microscope. Ultrathin, pulled borosilicate glass needles (outer diameter, 
50 µm) were used. The injections contained 1 to 10 µg labeled antibody and/or 50 µg labeled ovalbumin.
Intravital Microscopy
We have described our general methods in detail in a recent publication.8 During the microscopy, mice were anesthetized with 1 L/min of 1% to 2% isoflurane (Ohmeda, Liberty Corner, NJ) in oxygen and placed on the stage of a specially equipped epifluorescence microscope. Pupils were constricted with topical 4% pilocarpine to increase reproducibility and to maximize the area and the thickness of the iris, for clearer viewing. A viscous gel (Vidisic Gel; Mann Pharma, Berlin, Germany) was applied between the cornea and aqueous immersion objectives. Both the pilocarpine and the gel keep the cornea from drying during the procedure. Images were captured with a black-and-white camera (CF 8/4 NNIR; Kappa, Gleichen, Germany) or a color camera (CF11 DSP; Kappa) coupled to an image intensifier (Kappa) and recorded in super VHS quality with a computer (Power PC G4, 500 MHz; Apple, Cupertino, CA) running image capture software (Adobe Premier 5.1c) with a video board (Igniter; Aurora Video Systems, Shelby Township, MI). Except as noted, recordings were made at least 6 hours after injection of fluorescent protein to allow time for excess protein and the resultant background fluorescence to be cleared. Most recordings were made with a 20x objective with a 0.06-mm2 camera field of view.
Labeled cells in the recordings were generally counted off-line by technicians masked to the experimental protocol. For the data shown in Figures 2 and 3 , the labeled cells were counted through the microscope with a 20x objective and an eyepiece grid indicating a 0.25-mm2 field of view. The results are expressed as cells per square millimeter of iris.
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Wholemount Immunohistology
Anterior segments were dissected and kept overnight in 70% ethanol at 4°C. After washing with TBS (50 mM Tris [pH 7.5] and 150 mM NaCl), the tissues were treated sequentially with a peroxidase blocking reagent (Dako, Carpinteria, CA), a blocking solution of 2% rabbit serum, and then biotin-blocking reagent (Vector Laboratories, Burlingame, CA). The tissues were incubated with primary antibody (anti-F4/80 [1:100] or control rat IgG2b [1:100]) overnight at 4°C. Detection of the bound antibody was performed with an avidin-biotin complex kit (Vectastain ABC; Vector Laboratories) and red substrate (Nova; Vector Laboratories).
Statistical Analysis
The Student’s t-test was used to determine probabilities for changes in mean cell counts over time. Differences were considered statistically significant if P ≤ 0.05.
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In addition to antigen-specific antibody binding, these cells have the potential to be labeled by non-antigen-specific binding, as by Fc receptors, pinocytosis, or phagocytosis. To test for these possibilities, one control was to inject similarly labeled, isotype-matched irrelevant murine IgG2b. A few cells displayed some fluorescence but the number and intensity of labeled cells were clearly less than with the anti-MHC class II antibody (Fig. 1C) . A second control was to inject the labeled specific antibody simultaneously with a 100-fold excess of irrelevant antibody. The irrelevant antibody failed to block the specific staining (data not shown). Finally, coinjection of red ovalbumin and green anti-MHC class II antibodies revealed that only a subset of the cells labeled with ovalbumin were also positive for MHC class II and vice versa (Fig. 1D) . The results of these control experiments are all in accord with the conclusion that most of the cell-associated fluorescence from the anti-MHC class II antibody is due to antigen-dependent binding.
Antigen-presenting cells in the iris stroma have various phenotypes that not only depend on whether the cell is a macrophage or dendritic cell but also depend on the cell’s state of activation or maturity.4 5 6 To begin characterization of the cells that ingest ovalbumin, rat anti-mouse CD11b and F4/80 monoclonal antibodies were labeled with fluorescent dyes and injected into anterior chambers. The general pattern of fluorescence observed 6 and 24 hours later was similar to the network of dendriform cells seen with labeled ovalbumin. Irrelevant isotype-matched rat IgG2b
control antibodies were picked up by some cells, but as with the mouse IgG2b control antibodies, the number and intensity of labeled cells was less than that with the monoclonal antibodies. When care is taken to not overconjugate the antibodies (see the Methods section), the isotype control antibodies label only approximately 10 cells/mm2. Figure 1E shows an example of green fluorescence/anti-F4/80-labeled cells around blood vessels. This pattern is similar to that seen in immunostaining of iris in vitro (Fig. 1F) . The iris shown in Figure 1G is from a mouse that received an intravenous injection of FITC-dextran to label blood vessels and an anterior chamber injection of orange fluorescent conjugate-anti-CD11b. This higher-magnification view clearly shows orange fluorescence labeling of a dendriform cell abutting the outer surface of a blood vessel.
With simultaneous injection of ovalbumin with one label and antibodies with a second label, color fluorescence intravital microscopy was used to quantitate the number of cells that were labeled by either or both proteins at 6 and 24 hours after injection. As shown in Figure 2A approximately the same density of cells (116–148 cells/mm2) were labeled by antibodies to CD11b or F4/80 as by ovalbumin. Anti-MHC class II labeled slightly fewer cells. There was no significant difference in the density of cells labeled at 6 hours compared with those labeled at 24 hours. Despite the similarity of labeling patterns seen when looking at each protein separately, a clear distinction was readily apparent when focusing on cells that were double labeled. Whereas most of the cells labeled with anti-F4/80 were also positive for ovalbumin, less than a quarter of the anti-CD11b labeled cells were positive for ovalbumin (Fig. 2B) . Fewer MHC class II–positive cells were double labeled with ovalbumin at 24 hours after the ovalbumin/antibody injection (P = 0.049). There was a trend for more CD11b-positive cells with ovalbumin at 24 hours, but this difference is not statistically significant (P = 0.066). A complementary analysis shows that the percentage of ovalbumin-labeled cells that were also positive for CD11b increased (P = 0.022) and there was a trend toward a decrease in the percentage of ovalbumin-positive cells that were labeled with anti-MHC class II antibody (P = 0.059; Fig. 2C ). A subsequent experiment in which red conjugate-ovalbumin and green conjugate-anti-CD11c were coinjected into the anterior chambers indicated that most (95%) of the ovalbumin-labeled cells were also positive for another marker of myeloid cells, CD11c, at 6 hours (Fig. 1H) . In that experiment, only 24% of the ovalbumin-labeled cells were CD11c positive at 24 hours.
The preceding results suggest that the antibodies label different cell populations, even though they label cells with a similar distribution and morphology. As a further test of this conclusion, anterior chambers were injected simultaneously with red fluorescence-anti-CD11b and green fluorescence-anti-MHC class II. No more than 10% of the CD11b positive cells and 10% to 20% of the MHC class II–positive cells were double labeled when viewed by intravital microscopy (Fig. 3) . Some of these irises were dissected at the 24-hour time point and prepared for confocal microscopy ex vivo. The higher-resolution microscopic images clearly showed intermixing of the in vivo-labeled CD11b and MHC class II cells with only a few double-labeled cells (Fig. 1I) . These results are consistent with reports that macrophages do not express high levels of MHC class II proteins on their cell surfaces unless activated to present antigen. Once the cells are so activated, they would be expected to upregulate cell surface proteins, such as CD80 and CD86, for costimulatory signals as well. Indeed, when anterior chambers are injected with green fluorescence/anti-MHC class II and a mixture of red fluorescence-antibodies to CD80 and CD86, most of the MHC class II–positive cells were colabeled with the CD80/CD86 antibody mix (Figs. 1J 1K) , although there were differences in the relative intensities of individual cells.
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Discussion |
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Our second major finding is that fluorescently labeled antibodies to cell surface proteins injected into the anterior chamber of mouse eyes preferentially labeled cells in the iris that express those proteins and, thereby, allowed those cells to be identified and tracked over time. Antibodies with differing specificities reached and appropriately labeled different populations of cells in the iris. Furthermore, we were readily able to detect some cell surface markers in vivo, such as CD11c, CD80, and CD86, even though the same antibodies did not perform well with conventional immunohistology.14 Under optimized conditions, similarly tagged, species- and isotype-matched control antibodies labeled many fewer cells, with lower intensities and for a shorter period than the cell-surface-specific antibodies. However, if care is not taken to avoid excessive conjugation to the fluorescent dye, the antibodies may be ingested and label cells in the same pattern as ovalbumin. Although we have no reason to suspect that ovalbumin would be more susceptible to aggregation than antibodies, this occurrence would not alter our interpretation that ovalbumin is taken up by pinocytosis and/or phagocytosis and that properly prepared IgG given at a much lower dose preferentially binds exposed antigens.
These results also have implications for understanding the distribution of antibody within the eye. We were somewhat surprised to find that intracamerally injected antibody readily reached cells within the iris, but the mouse iris has a thickness of very few cells and does not have a complete epithelial covering. More recently, we have found that antibody injected into the living mouse eye is readily bound by cells in the cornea, retina, and choroid (Yang P et al., unpublished data, 2002), indicating extensive ocular distribution of a molecule with a molecular weight of approximately 150,000.
The ovalbumin-labeled cells have morphologic and antibody-binding characteristics of macrophages and dendritic cells. Nearly all the ovalbumin-associated cells bound the anti-F4/80 antibody, which is a marker for both macrophages and dendritic cells.15 A subset of the ovalbumin-labeled cells were also positive for MHC class II protein and one or both of the CD80 and CD86 costimulatory proteins. CD11b and CD11c are integrin subunits used to characterize cells of myeloid lineage. We found that at 6 hours after injection, most ovalbumin-labeled cells were CD11c+, one seventh were CD11b+, no more than 10% of the CD11b+ cells were also positive for MHC class II protein, and, conversely, 10% to 20% of the MHC class II–positive cells were also CD11b positive. These results indicate that a majority of the antigen-presenting cells in the iris that initially take up foreign protein is CD11b-CD11c+ and a minority are CD11b+CD11c+. At 24 hours, a higher percentage of the ovalbumin-labeled cells was CD11b+ and a lower percentage was CD11c+ and MHC class II+, indicative of a shift from dendritic cells to macrophages.
In these studies, the density of ovalbumin-labeled cells did not decrease from 6 to 24 hours after injection, even though colabeling with antibodies changed during this period. A variety of factors could account for this observation. Some of the cells may change their phenotype over time. The persistence of signal from labeled antibodies or ovalbumin may vary with the specific antibody or cell type. Some labeled cells may undergo apoptosis and be phagocytosed by cells with a different phenotype, which would then be labeled. Although it may be predicted that ovalbumin-laden dendritic cells would leave the iris to present the antigen to lymphocytes in a lymphoid tissue during this time frame, our data suggest that they either represented a small percentage of the labeled cells or were replaced by new cells that were labeled by residual ovalbumin in the tissue. Time-lapse recordings suggest that most of the ovalbumin-labeled cells remain in the iris, because these cells are only rarely seen traversing the extravascular tissue (manuscript in preparation).
The in vivo immunohistology technique described herein is clearly an important tool for studying cell kinetics and interactions. Although there is a hazard that the antibodies or labeled protein may alter cell function, proper controls can be included in the experimental design to control for any artifactual effect. A prior publication14 and our unpublished observation have found that the hamster antibodies for CD11c, CD80, and CD86 do not work well for immunohistochemical staining of mouse tissue sections, but we found that they give strong signals when used in vivo. The critical epitopes may be more accessible to the antibody in vivo or easily destroyed in vitro, although we have preliminary evidence that at least part of the in vitro staining limitation may be with the secondary antibody.
These studies demonstrate that a tissue, the iris, which should be relatively spared from participating in the immune response, nonetheless contains an abundance of cells with the potential ability to present antigen. This seems paradoxical because this portion of the eye should have no exposure to microbial products under ordinary circumstances, and the iris is bathed by aqueous humor that contains a number of soluble immunosuppressive factors.16 Although the iris was once thought to lack dendritic cells, more recent in vitro studies in many species including Homo sapiens supports our in vivo observations that iris is rife with antigen-presenting cells.14 17 18 By differential expression of cytokines and costimulatory cell surface molecules, dendritic cells may direct T cells to activate or suppress individual immune responses.5 Processing and presentation of self-antigens by dendritic cells is increasingly being recognized as a component of peripheral tolerance.19 20 Nevertheless, Steptoe et al.17 have shown that macrophages cultured from normal rat iris are effective antigen-presenting cells and are not inherently immunosuppressive. Future studies will determine whether the antigen-presenting cells in the immunosuppressive environment of the iris are programmed to suppress lymphocyte-mediated inflammatory responses.
This report describes a novel method and establishes the specificity of the staining. In vivo immunohistology is much more applicable to the eye than other tissues, such as heart or mesentery, because the transparent cornea allows visualization without the artifact of surgical trauma. Autolysis, hypoxemic injury, and fixation artifacts are all potentially confounders in conventional immunohistology. These confounders are avoided by studying cell-surface antigen expression in vivo rather than in vitro. In studies in progress, we are using this method to study the evolution of cell death in living, inflamed tissue over time,21 the movement of specific cell populations with time-lapse video microscopy, and the surprising distribution of intravitreously injected antibodies to cell surface markers.
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Footnotes |
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Submitted for publication June 10, 2002; revised October 1 and November 13, 2002; accepted November 21, 2002.
Disclosure: M.D. Becker, None; S.R. Planck, None; S. Crespo, None; K. Garman, None; R.J. Fleischman, None; P. Dullforce, None; G.W. Seitz, None; T.M. Martin, None; D.C. Parker, None; J.T. Rosenbaum, 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: Stephen R. Planck, Casey Eye Institute, Oregon Health & Science University, 3375 SW Terwilliger Boulevard, Portland, OR 97239; plancks{at}ohsu.edu.
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