Inflammation, Vol. 31, No. 2, April 2008 ( # 2007) DOI: 10.1007/s10753-007-9055-x
Characterizing Extravascular Neutrophil Migration In Vivo in the Iris Stephen R. Planck,1,2,3 Matthias D. Becker,1,5 Sergio Crespo,1 Dongseok Choi,4 Kellen Galster,1 Kiera L. Garman,1 Rainer Nobiling,5 and James T. Rosenbaum1,2,3
Abstract—Extravascular neutrophil migration is poorly characterized in vivo. To test the hypothesis that this migration is a non-random process, we used videomicroscopy to monitor neutrophils in irises of living mice with endotoxin-induced uveitis (EIU). Paths of individual cells were analyzed. Nearly all of these cells were moving in divergent directions, and mean displacement plots indicated that the predominant movement was random. The paths of some cells were fit to bivariate autoregressive integrated moving average models that revealed at least two modes of movement: random search and linear trend. Cell speed was significantly reduced by the actin inhibitor, cytochalasin D. The pattern of migration for neutrophils is in marked contrast to what we previously described for antigen-presenting cells in the iris, but somewhat resembles recent descriptions for T cells within a lymph node. Characterization of extravascular migration of neutrophils has important implications for understanding infection and immunity. KEY WORDS: intravital microscopy; leukocyte trafficking; uveitis.
The neutrophil is a highly mobile, phagocytic leukocyte that is consistently among the first to arrive at a site of infection or inflammation. The absence of neutrophils [1] or impairment in their motility [2] leads to recurrent or overwhelming infection. More recently, neutrophils have been recognized for their role in regulating the response of T cells and adaptive immunity
[3]. A great deal is known about the behavior of neutrophils including their rolling along the inside wall of a vessel wall and their diapedesis across the vascular endothelium. These processes are controlled by a variety of chemotactic factors and adhesion molecules [4, 5]. In contrast, virtually nothing is known about the extravascular migration of neutrophils, which is too slow to be appreciated by real-time videomicroscopy. It seems plausible, however, to hypothesize that this locomotion is non-random or directed. Several laboratories have recently reported novel time-lapse imaging of T cell or dendritic cell migration within the lymph node [6–8]. Somewhat surprisingly, these studies indicate that these leukocytes mostly follow a pattern of random walk. These observations of cell trafficking patterns and cell–cell interactions are changing the understanding of the immune response. The eye is especially suited for studying leukocyte migration because the translucent cornea allows microscopic imaging of tissues behind it without the need to resort to surgical trauma to expose this tissue. We have previously used time-lapse photography to characterize
Electronic supplementary material The online version of this article (doi:10.1007/s10753-007-9055-x) contains supplementary material, which is available to authorized users. Planck and Becker contributed equally. 1
To whom correspondence should be addressed at Department of Ophthalmology, Casey Eye Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. E-mail:
[email protected] 2 Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, OR, USA. 3 Department of Medicine, Oregon Health & Science University, Portland, OR, USA. 4 Department of Public Health & Preventive Medicine, Oregon Health & Science University, Portland, OR, USA. 5 Present Address: Universität Heidelberg, Heidelberg, Germany.
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the movement of phagocytic cells in the murine iris and found that these cells were surprisingly relatively immobile [9]. The local injection of bacterial endotoxin produces an acute inflammatory response within the murine eye. The early response is predominantly neutrophilic [10]. Using time-lapse videomicroscopy, we tested the hypothesis that neutrophil migration within the extracellular matrix of mouse iris would be primarily directed or constrained. We demonstrate that the neutrophils within the iris stroma are in near constant motion with elements of linear transitions and random walks.
METHODS Animals Female BALB/c mice (Jackson Laboratories, Bar Harbor, ME) were 6 to 8 weeks old at the time of the experiments. All procedures were in accordance with the ARVO Animal Statement and the NIH Guiding Principles in the Care and Use of Animals. Time-Lapse Microscopy To minimize eye movement during the recording sessions, mice were anesthetized with 600 μg xylazine and inhaled isoflurane (0.5 to 2%) in oxygen and their heads were braced. Pupils were constricted with topical 4% pilocarpine. A digital video system [11] was used to record intravital microscopy images in time-lapse mode. To minimize light exposure to the eyes, so as to not bleach the fluorescent dye or cause phototoxicity, a computer-controlled shutter opened 0.8 s before the frame was to be captured and closed immediately afterwards. To compensate for eye movement and motion artifacts from breathing and heartbeat of the animal, the frames need to be aligned. For the first series of videos (Figs. 1, 2 and 3), frame registration was done with VISAR image stabilization software by the Solar Physics Group at the NASA Marshall Space Flight Center, Huntsville, AL. For the second series of videos (Figs. 4, 5 and 6), we used Image Pro Plus 5.0 (Media Cybernetics, Bethesda, MD)
Fig. 1. Frame from time-lapse movie of an iris in a mouse after intravitreal endotoxin injection. The refractive dots (arrows) are infiltrating leukocytes, predominately neutrophils. The lines indicate the paths covered by 13 representative cells during 81 min with triangles showing the position of each cell at the beginning of the recording. The letters are cell designations also employed in Fig. 3b and the text.
human serum albumin (Baxter Healthcare, Glendale, CA) [11]. In these animals, intravascular leukocytes were fluorescently labeled by intravenous injection of rhodamine 6G (35 mg/kg; Sigma Chemicals, St. Louis, MO) 10 min before microscopy and infiltrating leukocytes were seen by their refraction of light from rhodamine fluorescence. Time-lapse recordings were made at three frames per minute for 90 min beginning 6 h after injection of endotoxin. Quantification of Cell Migration The NIH image program (Version 1.61, US National Institutes of Health) was used to record the X–Y coordinates of individual cells in each frame of the recordings. The coordinates were then used to calculate the direction and speed (μm/min) of cell movement and to plot the path of single cells. Mean displacement plots were created in order to characterize the migratory pattern as directed, random, or confined [12]. The patterns of movement were further analyzed by fitting bivariate autoregressive integrated moving average models to cell coordinates [13–15].
Endotoxin-Induced Uveitis
Cytochalasin D Treatment
Uveitis was induced by intravitreal injection of 2 μl saline containing 250 ng E. coli 055:B5 endotoxin (List Biological Laboratories, Campbell, CA) and 0.25%
For this series of experiments, mice received 4 μg LPS intravitreally at time 0. Starting at 4 h postendotoxin injection, time-lapse videos were recorded for
Time-Lapse Intravital Microscopy of Iris
Fig. 2. Histogram of speeds of 13 cells measured in 20-s intervals for a total of 81 min.
Fig. 3. Mean displacement plot. Averaging the data from all 13 cells results in a nearly linear plot indicating overall randomness to their movement.
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Fig. 4. Cytochalasin D treatment significantly (p=0.035) lowers the speed of infiltrating cells.
Fig. 5. Cytochalasin D treatment does not alter the predominantly random migration pattern as indicated by nearly linear mean displacement plots.
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Fig. 6. Neither DMSO vehicle nor the time point of recording is responsible for the slower speed seen in the cytochalasin D treated eyes.
1 h. At 5.5 h post-endotoxin injection, the mice received 2 μl intravitreal injections containing 100 ng cytochalasin D [16, 17] or vehicle control (DMSO). Video recordings were initiated immediately after treatment, and cell migration was tracked for an additional 1 to 2 h. For each video, at least 12 cells were randomly chosen by a masked observer and tracked as described above. A mixed effects model was used to test the differences in speeds between two groups of each data set while accounting for potential correlations between cells within a movie and a mouse.
RESULTS Six hours after intravitreal injection of endotoxin, the iris stroma was filled with invading leukocytes, predominately neutrophils and some macrophages[10, 18, 19]. Time-lapse videos show that nearly all of the infiltrating cells were in motion (see “Electronic supplementary material” for representative video). Although some cells paused, very few infiltrating cells were completely immobile during the recording period. We have observed this pattern of migration in more than 15 independent studies and chose arbitrarily to analyze the cell movement most extensively in the initial video that we obtained. Frame-by-frame determination of the X–Y coordinates of individual cells allowed us to plot their paths and calculate their velocities. Since an analysis of an
individual cell’s movement path is laborious, representative cells were chosen for detailed study. Cells whose migratory patterns typified those of other cells were chosen by viewing the video dozens of times. Figure 1 shows a frame from the movie overlaid with the paths of 13 representative cells (see “Electronic supplementary material” for color figure). The histogram in Fig. 2 is a compilation of the speeds of these cells for 20-s intervals over the course of 81 min. The mean speed was 7.6± 4.7 μm/min and the maximum speed of individual cells in 20-s intervals ranged from 21 to 44 μm/min. Visual inspection of the cell paths shown in Fig. 2 reveals at least two general patterns of movement. For one, the cells appear to roam around a given location and for the other, the cells move in a more linear fashion. The linear movements often, but not always, appear to parallel blood vessels. Some of the paths appear to circumscribe vessels. A given cell may follow one pattern transiently and then switch to the other pattern. Two cells may come together and then move away in different directions suggesting the possibility of attractive and repulsive forces at work. We used two different methods to analyze the cell movements mathematically. One was to create mean displacement plots similar to those used to analyze cell movements within a lymph node [12]. The other was to test the fit of the cell paths to bivariate autoregressive integrated moving average (ARIMA) models, which are widely used in time series analyses [13–15]. When the data from all cells are combined and plotted as a single series in a mean displacement plot, the resulting line is nearly linear (Fig. 3). This indicates that the combined movement of all cells is consistent with random walks. ARIMA models were fit to the paths of three cells and had p
