In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

In Vivo Imaging of Leukocyte Trafficking in the Steady State.

We used 2P microscopy to examine leukocyte trafficking in LysM-GFP mice (14), in which endogenous neutrophils are brightly labeled and monocytes and macrophages are labeled to a lesser extent (15). For imaging, mice were anesthetized, intubated, and placed on a ventilator. The left lung was exposed by performing a lateral thoracotomy, and the animal was placed in a custom 2P imaging chamber. To visualize the pulmonary vasculature and determine whether neutrophils were extravascular, we injected quantum-dots (Q-dots; Invitrogen) (Fig. 1A). We first examined lungs in healthy mice by using intravital 2P microscopy and found that many neutrophils were sequestered in the pulmonary microcirculation (Movie S1) as reported by others (16). In addition to the circulating pool of neutrophils, we found a substantial number of extravascular neutrophils in the lung (Fig. 1A, yellow arrowheads). To address the possibility that the imaging preparation itself induced neutrophil extravasation, we examined freshly explanted lungs from healthy mice and found that they also contained a population of extravascular neutrophils (Fig. 1B). In this respect, the lung resembled secondary lymphoid organs such as lymph nodes (Fig. 1C), which contained tissue-resident neutrophils, but not other nonlymphoid tissues including heart, brain, liver, kidney, small bowel, and footpad (Fig. 1 D–I), which were virtually free of extravasated neutrophils.

Intravital imaging and single-cell tracking revealed that extravascular neutrophils were weakly motile (mean velocity = 2.88 μm/min) (Fig. 2 A–D), and the number of motile cells varied widely from mouse to mouse (Movie S1 and Movie S2). In contrast, neutrophils in lung explants were predominantly motile and migrated randomly through the tissue with a mean velocity of 8 μm/min (Fig. 2 E–H and Movie S3), which is similar to interstitial velocities reported for neutrophils at sites of inflammation (17).

Impact of Inflammation on Leukocyte Recruitment and Motility.

One possible explanation for the variability of neutrophil motility in vivo is that perhaps individual mice in our colony had occult respiratory infections or inflammation that could affect cell motility. Moreover, the robust neutrophil motility observed in the explanted lungs could be due to the trauma associated with surgical removal of the lung and ex vivo imaging. To test whether inflammation influenced neutrophil motility in the lung, we administered bacteria intratracheally and assessed pulmonary neutrophil motility by intravital 2P microscopy. Within minutes of bacterial challenge, we observed a dramatic influx of cells from the circulation and a significant increase in resident neutrophil motility (mean velocity = 9.68 μm/min) (Fig. 2 I–L and Movie S4). Similar results were obtained after intratracheal administration of Escherichia coli BioParticles (Movie S5). In response to bacterial infection, neutrophils in the lung often formed dynamic clusters (Fig. 3 A and B and Movie S6) reminiscent of leukocyte behavior observed in other tissues after infection (15, 18, 19).

To determine whether cell behaviors were specific to bacterial challenge or representative of inflammation in general, we imaged neutrophil recruitment during lung transplant-mediated ischemia reperfusion injury (20), a process that contributes to high rates of early and late graft dysfunction in the clinics (21). This model is advantageous because ischemic injury in lung grafts is associated with robust neutrophil recruitment (3). Furthermore, by using LysM-GFP mice as recipients, but not donors, we could image exclusively neutrophils originating from the circulation. We observed a substantial recruitment of GFP^high cells to the lung graft 2 h after transplantation into LysM-GFP recipients (Fig. 3 C and D). Flow cytometric analysis demonstrated that ≈95% of the GFP^high lung-infiltrating cells, with a high degree of side scatter, express high levels of Gr1, Ly6G, and CD11b, and do not express CD115, indicating that these cells are primarily neutrophils (Fig. S1). Ischemia reperfusion injury induced vigorous neutrophil extravasation as well as cell arrest in the subpleural capillary network (Fig. S2). Extravascular neutrophils in the graft migrated with robust motility similar to neutrophils responding to bacterial challenge or those tracked in explanted lungs (Fig. 2). Excluding stationary cells in the clusters, the mean neutrophil velocity (8.85 μm/min) was significantly higher after ischemia reperfusion injury than in baseline lungs (2.88 μm/min). Similar to lungs challenged with bacteria, transplanted lung grafts contained large neutrophil clusters (Fig. 3 C and D and Movie S7). Both the frequency and size of neutrophil clusters were significantly increased over intravital baseline lungs (Fig. 3E). Moreover, many neutrophil clusters in transplanted lungs were dynamic, increasing and decreasing in size during our 30- to 60-min imaging window (Movie S7).

In Vivo Tracking of Blood Monocytes with Nontargeted Q-Dots.

In addition to GFP-labeled neutrophils, we also saw a population of red cells in the circulation that had the same emission characteristics as the 655-nm Q-dots used to label the blood vessels (Fig. S3A). Using flow cytometry, we found that the Q-dot-positive cells expressed F4/80, the M-CSF receptor (CD115), the myeloid marker CD11b, Ly-6C, and low levels of Gr1 (Fig. S3B), which is consistent with these cells being peripheral blood monocytes (22, 23). Within the lung, we typically observed Q-dot-positive monocytes rocketing through large vessels and moving in a jerky intermittent pattern in alveolar capillaries and the subpleural capillary network. In the absence of inflammation, these monocytes rarely arrested inside vessels or entered the tissue. However, under inflammatory conditions, Q-dot-positive monocytes were found rolling in the vessels and occasionally entering the tissues at 2–3 h after transplantation (Movie S8).

Neutrophil Clustering Is Dynamic, Transient, and Associated with Q-Dot-Positive Cells.

The observation that transplanted lungs contained dynamic neutrophil clusters is surprising, because the graft would be expected to be more uniform in terms of ischemia-induced neutrophil recruitment signals than infected lungs where bacteria are restricted to specific regions of the tissue. This finding suggested that neutrophils were responding to important local migration cues, perhaps originating from a cellular source in the engrafted lung. In support of this hypothesis, we observed individual Q-dot-labeled monocytes leaving the circulation (Fig. 4A and Movie S8) and colocalizing with large neutrophil clusters (50–100 μm below the pleura). Notably, we found that 60.9% of neutrophil clusters are closely associated with at least one Q-dot-labeled monocyte (Fig. S4). Of the neutrophil clusters that are associated with Q-dot-labeled cells, 78.6% are associated with one monocyte, 14.3% with two monocytes, and 7.1% with three monocytes. We created a “heatmap” of cell density and speed to visualize cell migration behavior over time in the lung graft. This approach generated a spatiotemporal map of cell chemotaxis in vivo and allowed us to test whether neutrophil distribution was regulated by stable global signals or by transient local signals. Using this analysis tool, we found that the migration behavior of neutrophils was nonuniform and consistent with cells responding to spatiotemporally restricted chemotactic signals in the graft (Fig. 4 B and C and Movie S9), rather than randomly distributing through the tissue as expected for global recruitment signals. At local regions where clusters began to form, neutrophil velocity in the vicinity increased and the cell tracks were relatively straight. Neutrophils joining clusters showed a significant forward bias (Fig. 4D) with increased velocity as they approached within 50 μm of the cluster center (8.59 μm/min) and cell tracks displayed high meandering coefficients (>0.8) consistent with chemotaxis (Fig. 4 E and F). By contrast, cells at a distance of tens of microns from the center of the clusters moved more slowly (4.15 μm/min) and had lower meandering coefficients as expected for random migration (Fig. 4 G and H). Chemoattraction was short-lived, and neutrophils could be found leaving mature clusters after 10–20 min. These cells were often recruited to nascent clusters forming nearby, indicating that they retained their chemotactic potential. In most cases, clusters did not completely disassociate over our imaging time window and a number of neutrophils typically remained at the cluster center as it decreased in size.

Clodronate-Liposome Depletion Inhibits Neutrophil Extravasation Downstream of Endothelial Arrest.

The physical association of Q-dot-positive cells with neutrophil clusters suggested that monocytes might regulate neutrophil chemotaxis transiently and locally at effector sites in the lung tissue. To assess the role of monocytes in neutrophil chemotaxis and cluster formation, we used repeated i.v. injections of clodronate liposomes (22) to deplete monocytes in recipient mice before lung transplantation. Compared with control conditions, clodronate-liposome treatment resulted in reduced neutrophil cluster formation in lung tissue at 2 h after transplantation (Fig. 5 A and B). Unexpectedly, we also observed a corresponding decrease in the number of extravascular neutrophils at this time (Fig. 5B). Upon closer examination, 2P microscopy revealed a striking defect in neutrophil transendothelial migration. Neutrophils accumulated inside vessels, clogging arterioles as well as capillaries. Despite the fact that numerous neutrophils firmly arrested along the endothelium, the majority of cells failed to show evidence of diapedesis during our 2-h imaging window. In control transplant recipients <5% of neutrophils were intravascular at 2-h after engraftment, in contrast to clodronate-liposome–treated mice, where >90% of neutrophils remained trapped within vessels (Fig. 5C).

Mice and Monocyte Depletion.

BALB/c mice were purchased from The Jackson Laboratories. LysM-GFP mice were obtained from Klaus Ley (La Jolla Institute for Allergy and Immunology, La Jolla, CA) and maintained at our facility. Escherichia coli (K-12 strain) BioParticles conjugated with tetramethylrhodamine (Invitrogen) (500 μg/mL) or 100,000 cfu of L. monocytogenes were administered intratracheally after dilution in 50 μL of PBS. Clodronate liposome suspensions were prepared as described (31). Monocytes were depleted by treating mice with three doses of i.v. clodronate-containing liposomes given 48 (200 μL), 24 (100 μL), and 6 h (100 μL) before lung transplantation.

Lung Transplantation.

After 18-h storage in low-potassium dextran glucose at 4 °C, left BALB/c lungs were transplanted orthotopically into LysM-GFP mice as described (20, 36).

Two-Photon Microscopy.

Time-lapse imaging was performed with a custom built 2P microscope running ImageWarp acquisition software (A&B Software). Mice were anesthetized with an i.p. injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and maintained with halved doses administered every hour. Mice were intubated orotracheally with a 20 G angiocatheter and ventilated with room air at a rate of 120 breaths/min and with a tidal volume of 0.5 mL. The left lung was exposed through a left thoracotomy, and the lung was imaged by using a custom built chamber maintained at 37 °C. A small ring of VetBond was used to attach the lung tissue to the bottom of the cover glass without exerting pressure directly on the lung. For time-lapse imaging of leukocyte migration in the tissue parenchyma, we averaged 15 video-rate frames (0.5 s per slice) during the acquisition to match the ventilator rate and minimize movement artifacts. Each plane represents an image of 220 × 240 μm in the x and y dimensions. Twenty-one to 31 sequential planes were acquired in the z dimension (2.5 μm each) to form a z stack. To visualize blood vessels, 20 μL of 655-nm nontargeted Q-dots in 100 μL of PBS were injected i.v. Two-photon excitation produces a second harmonic signal from collagen (9) around alveoli, thus providing a useful landmark for the air spaces. Explanted tissue was examined as described (8, 9).

Flow Cytometry.

Whole blood for analysis of Q-dot–positive cells and lung digests for analysis of GFP^high cells were prepared as described (37). Cells were first incubated with anti-mouse CD16/CD32 for 15 min at 4 °C to block Fc receptors (Fc γ III/II receptor, 2.4G2; BD Pharmingen). Cells were stained with fluorochrome-labeled anti-F4/80 (clone BM8), anti-Ly-6C (clone AL-21), anti-CD11b (clone M1/70), anti-CD115 (clone AFS98), anti-Ly-6G (clone 1A8), and anti-Gr-1 (clone RB6-8C5) antibodies or isotype controls (BD PharMingen). Analysis was performed on a FACSCalibur (BD Bioscience) equipped for four-color flow cytometry.

Data Analysis.

Multidimensional rendering was done with Imaris (Bitplane), whereas manual cell tracking was done by using Volocity (Improvision). Data were transferred and plotted in GraphPad Prism 5.0 (Sun Microsystems) for the creation of the graphs. The neutrophil cluster analysis was performed by using the cluster analysis function of the T cell Analysis program (TCA; John Dempster, University of Strathclyde, Glasgow, Scotland) using a 25-μm radius threshold for cell-to-cell distances. Pseudocolored heatmaps were created to visualize neutrophil speed [0 μm·min⁻¹ (purple) to 12 μm·min⁻¹ (red), and density (0–256 gray from purple to white)] within the tissue volume. Neutrophil speed and density were considered to be continuous functions and the fraction of neutrophils, which are imaged, as samples of this underlying distribution. These neutrophils were used to create a kernel estimate of the complete distribution, and the final video is color-coded based on this speed or density estimate. The kernel speed and density estimate uses the Parzen window approach with a Gaussian Kernel (38). The Gaussian Kernel was chosen by hand. The unpaired two-tailed Student's t test was used for statistical analysis.