In vivo two-photon imaging reveals monocyte-
dependent neutrophil extravasation during
Daniel Kreisela,b,1, Ruben G. Navab,1, Wenjun Lib,1, Bernd H. Zinselmeyera,c,1, Baomei Wanga, Jiaming Laib,
Robert Plessd, Andrew E. Gelmana,b, Alexander S. Krupnickb, and Mark J. Millera,2
Departments ofaPathology and Immunology,bSurgery, anddComputer Science and Engineering, Washington University, St. Louis, MO 63110; andcNational
Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20824
Edited* by Michael D. Cahalan, University of California, Irvine, CA, and approved September 13, 2010 (received for review June 21, 2010)
Immune-mediated pulmonary diseases are a significant public
health concern. Analysis of leukocyte behavior in the lung is
essential for understanding cellular mechanisms that contribute to
normal and diseased states. Here, we used two-photon imaging to
study neutrophil extravasation from pulmonary vessels and sub-
sequent interstitial migration. We found that the lungs contained
a significant pool of tissue-resident neutrophils in the steady state.
In response to inflammation produced by bacterial challenge or
transplant-mediated, ischemia-reperfusion injury, neutrophils were
rapidly recruited from the circulation and patrolled the interstitium
and airspaces of the lung. Motile neutrophils often aggregated in
dynamic clusters that formed and dispersed over tens of minutes.
These clusters were associated with CD115+F4/80+Ly6C+cells that
had recently entered the lung. The depletion of blood monocytes
but acted by inhibiting neutrophil transendothelial migration up-
stream of interstitial migration. Our results suggest that a subset
of monocytes serve as key regulators of neutrophil extravasation
in the lung and may be an attractive target for the treatment of
inflammatory pulmonary diseases.
lung|two-photon microscopy|transendothelial migration|ischemia|
agents. Specifically, neutrophil-mediated responses in the lung are
critical as a first line of defense against infections (1). To this end,
a recent study demonstrated that respiratory influenza infection of
neutrophil-depleted mice is associated with enhanced viral repli-
cation, severe pulmonary inflammation, and death (2). However,
inotherdisease processessuch aspulmonaryischemiareperfusion
injury, neutrophil activation can be deleterious (3). Inhibition of
neutrophil recruitment has proven beneficial in several experi-
mental models of lung inflammation (4). Thus, a better un-
derstanding of neutrophil trafficking in the lung is crucial for the
development of new and effective therapeutic strategies.
Two-photon (2P) microscopy has been widely adopted by
immunologists and microbiologiststo study single-cell dynamics in
tissueexplantsand living mice(5–7). Recently,weandothershave
used 2P microscopy to study cellular immune responses in
explanted lungs (8–11). Two-photon microscopy has superior
spatiotemporal resolution to positron emission tomography and
magnetic resonance imaging (12, 13), and greater tissue penetra-
tion and less photodamage, than confocal microscopy. Despite
the potential advantages of using 2P microscopy in pulmonary
research, this approach has not been applied to study leukocyte
dynamics in vivo due to the technical difficulties of imaging a rap-
idly ventilated lung.
Here, we used 2P time-lapse imaging to study neutrophil traf-
ficking in the lungs of mechanically ventilated mice. We observed
a significant pool of lung-resident neutrophils in the steady state.
In response to inflammation, neutrophils were recruited rapidly
ungs are thesite of many human diseases, whichis in large part
due to their constant exposure to many infectious and noxious
from the circulation and displayed robust interstitial migration in
the lung. Motile neutrophils formed dynamic clusters around re-
cently emigrated quantum-dot (Q-dot) positive cells that express
monocyte surface markers. Depleting blood monocytes with
clodronate liposomes reduced neutrophil clustering in the lung by
impairing neutrophil transendothelial migration, which left large
numbers of neutrophils stranded along the vascular endothelium.
Our results suggest that blood monocytes play a central role in
regulating neutrophil trafficking in the lung.
In Vivo Imaging of Leukocyte Trafficking in the Steady State. We
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
(Q-dots; Invitrogen) (Fig. 1A). We first examined lungs in healthy
mice by using intravital 2P microscopy and found that many neu-
trophils were sequestered in the pulmonary microcirculation
(Movie S1) as reported by others (16). In addition to the circu-
lating pool of neutrophils, we found a substantial number of ex-
travascular neutrophils in the lung (Fig. 1A, yellow arrowheads).
To address the possibility that the imaging preparation itself in-
duced neutrophil extravasation, we examined freshly explanted
lungs from healthy mice and found that they also contained
a population ofextravascular 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 othernonlymphoid tissues includingheart, brain,liver,kidney,
small bowel, and footpad (Fig. 1 D–I), which were virtually free of
Intravital imaging and single-cell tracking revealed that extra-
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 mi-
grated randomly through the tissue with a mean velocity of 8 μm/
Author contributions: D.K., R.G.N., W.L., B.H.Z., B.W., J.L., A.E.G., A.S.K., and M.J.M. de-
signed research; D.K., R.G.N., W.L., B.H.Z., B.W., J.L., A.E.G., A.S.K., and M.J.M. performed
research; R.P. contributed new reagents/analytic tools; R.G.N., B.H.Z., B.W., R.P., and
A.E.G. analyzed data; and D.K., R.G.N., and M.J.M. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1D.K., R.G.N., W.L., and B.H.Z. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 19, 2010
| vol. 107
| no. 42
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 re-
spiratory infections or inflammation that could affect cell motility.
Moreover, the robust neutrophil motility observed in the explan-
ted lungs could be due to the trauma associated with surgical
removal of the lung and ex vivo imaging. To test whether in-
flammation influenced neutrophil motility in the lung, we admin-
motility by intravital 2P microscopy. Within minutes of bacterial
challenge, we observed a dramatic influx of cells from the circu-
lation 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
challengeor 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
tion in various tissues of LysM-GFP mice. Neu-
trophils (bright green) and macrophages (dim
green) are easily distinguishable based on their
different brightness levels and distinct morpho-
logical characteristics. Blood vessels (red) were
labeled by i.v. injection of nontargeted 655-nm
Q-dots and the laser-induced second harmonic
generation signal appears blue. In addition to
there is a large number of extravascular tissue-
resident neutrophils (yellow arrowheads) seen in
lungs in vivo (A) (Movie S1) and explanted lungs
(B), and in lymph nodes (C). In heart tissue (D),
resident macrophages were observed, but neu-
trophils were present only within blood vessels.
(Scale bar: 80 μm.) Lower are zoomed views of
Upper. (Scale bar: 20 μm.) Tissue-resident mac-
rophages (white arrowheads) are found in other
tissues, but neutrophils (yellow arrowheads) are
detected primarily within the vasculature in
brain (E), liver (F), kidney (G), small intestine (H),
or hind footpad (I). (Scale bar: 15 μm.)
Neutrophil and macrophage distribu-
tissue in vivo, ex vivo, and under inflammatory conditions.
(A) Intravital 2P imaging of resident neutrophils (green) in
the parenchyma of the lung in vivo. Images are individual
frames from a continuous time-lapse movie (Movie S2). A
rare motile cell (yellow arrowheads) is shown migrating
through the interstitial tissue. (B) Individual cells were
tracked and cell displacement squared (μm2) vs. time (min)
shows a strong linear correlation indicative of random cell
migration. Plots of average neutrophil track speed (C) and
meandering index (MI), n = 20 (D). The MI was calculated
by dividing the distance a cell traveled from its starting
point by the track length. Values of >0.8 are commonly
associated with chemotaxis, whereas values of <0.5 are
consistent with random cell migration. (E) Neutrophils
(green) migrating in explanted lung tissue (Movie S3). A
representative neutrophil track is highlighted (yellow ar-
rowhead). Neutrophil displacement squared vs. time plot
(F), mean track speed (G), and MI (H) in lung explants, n =
20. (I) Neutrophil (green) behavior in vivo 5 min after
cytogenes, EGD strain) (Movie S4). A representative neu-
trophil track is highlighted (yellow arrowhead). Neutrophil
displacement squared vs. time plot (J), mean track speed
(K), and MI (L) after bacterial challenge, n = 16. (Scale bars:
10 μm.) Relative time is displayed in min:sec.
Time-lapse imaging of neutrophil behavior in lung
| www.pnas.org/cgi/doi/10.1073/pnas.1008737107 Kreisel et al.
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 ob-
served a substantial recruitment ofGFPhighcells tothelung graft2
Flow cytometric analysis demonstrated that ≈95% of the GFPhigh
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). Is-
chemia reperfusion injury induced vigorous neutrophil extravasa-
tion as well as cell arrest in the subpleural capillary network (Fig.
S2). Extravascular neutrophils in the graft migrated with robust
motilitysimilar to neutrophils responding to bacterial challengeor
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
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 character-
istics 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 mono-
cytes rocketing through large vessels and moving in a jerky in-
termittent pattern in alveolar capillaries and the subpleural
capillary network. In the absence of inflammation, these mono-
cytes 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 con-
tained 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
bacteriaare restrictedtospecific regions ofthetissue.Thisfinding
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 in-
dividual 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
tospatiotemporally restricted chemotacticsignals inthegraft(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.
Neutrophilsjoiningclustersshoweda significantforward bias(Fig.
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 mi-
gration (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 cellswithneutrophilclusters suggestedthatmonocytes
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 trans-
plantation (Fig. 5 A and B). Unexpectedly, we also observed a cor-
responding decrease in the number of extravascular neutrophils
photon images of neutrophil (green) distribution 5 (A) and 30 min (B) after
intratracheal administration of bacteria (Movie S6). Nontargeted Q-dots (red)
were injected i.v. 30 min before bacterial challenge. Neutrophilsin theclusters
were often nonmotile. (Scale bar: 60 μm.) Two-photon images of neutrophil
(green) distribution in lung grafts 2 h after transplant (C) and 2.5 h after
transplant (D) (Movie S7). Time stamp is shown in min:sec. Yellow arrowheads
show clusters that are forming or remain similar in size; white arrowheads
show clusters that appear to dissociate. (E) The number of neutrophils per
cluster in steady-state lungs (gray squares), lung explants (red triangles), and
lungs after transplantation (blue diamonds) (*, 0.0371; **, 0.0088).
Neutrophils cluster after intratracheal bacterial challenge. Two-
Kreisel et al. PNAS
| October 19, 2010
| vol. 107
| no. 42
at this time (Fig. 5B). Upon closer examination, 2P micros-
copy revealed a striking defect in neutrophil transendothelial
migration. Neutrophils accumulated inside vessels, clogging arteri-
oles as well as capillaries. Despite the fact that numerous neu-
trophils firmlyarrested along the endothelium, the majority ofcells
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).
Previous in vivo imaging studies of the lung using the adoptive
transfer of fluorescently or radioactively labeled neutrophils pro-
vided important anatomical and physiological insight (16). How-
and for the most part, imaging was restricted to superficial vessels.
Moreover, there is concern that the adoptively transferred cell
populations might traffic abnormally due to their ex vivo prepara-
tion. Importantly, our approach allowed the trafficking of endog-
enous neutrophils and monocytes to be analyzed quantitatively
both in the microcirculation and as they migrate through the pa-
in vivo description of neutrophil migration in pulmonary tissue.
Our approach took advantage of LysM-GFP mice (14), in which
endogenous neutrophils are brightly labeled and monocytes and
macrophages are labeled to a lesser extent (15). Others have
macrophages after lymphocytic choriomeningitis virus infection
(24). However, similar to the conclusions of others (15), our flow
cytometric analysis of the lung confirmed that the brightest in-
filtrating GFP+cells were neutrophils (Fig. S1).
We found that the lungs contain a significant pool of tissue-
resident neutrophils in the steady state. This was similar to lym-
few if any extravascular neutrophils. Lung-resident neutrophils
of the lung against infection or alternatively these cells could
contribute to the lung’s susceptibility to chronic inflammatory
diseases. Lung infiltrating neutrophils are likely to contribute to
the comparatively high rates of ischemia reperfusion injury-me-
diated lung graft dysfunction (25). The presence of neutrophils in
the lung parenchyma might represent an evolutionary tradeoff in
threshold for inflammatory disease.
Through serendipity, we found that i.v.-injected nontargeted Q-
dots fluorescently labeled monocytes in the peripheral blood. The
most likely explanation forthis phenomenonis that monocytesare
labeled byingestingQ-dotsinthecirculation similartoestablished
protocols using fluorescent latex beads (23, 26). The Q-dot–
labeling approach could provide a facile method to study mono-
such as atherosclerosis or arthritis. Q-dot labeling has the advan-
tages that cells are labeled in vivo (i.e., without ex vivo manipu-
chemia reperfusion injury. (A) Blood vessels (red) were labeled
by i.v. injection of nontargeted 655-nm Q-dots. A time-lapse
2P image sequence shows a Q-dot–positive cell leaving the
pulmonary vasculature through a small branch of a medium-
sized vessel (yellow track). Extravasation of the Q-dot–positive
cell is associated with neutrophil extravasation and sub-
sequent cluster formation (Movie S8). (Scale bar: 20 μm.) A
heatmap visualization was generated to show the spatio-
temporal changes in neutrophil velocity (B) and density (or
integrated intensity) (C) (Movie S9). For speed, the color scale
ranges from <2 μm/min (blue) to >10 μm/min (red). A local
increase in cell velocity (B) (white arrowheads) precedes a 4-
fold increase in cell density (yellow arrows) (C). (Scale bar: 60
μm.) (D) Neutrophil tracks (yellow arrows) show a symmetrical
short-range migration bias toward the cluster. (Scale bar: 15
μm.) Time stamps in A–D show relative time in min:sec. Neu-
trophils were divided in two groups based on distance from
the center of the cluster, and the migration of each group was
analyzed separately. Mean track speed (E) and MI (F) of
neutrophils approaching within 50 μm of clusters, n = 13.
Mean track speed (G) and MI (H) of neutrophils distal to
clusters (>50 μm from clusters), n = 13.
Leukocyte dynamics during transplant mediated is-
| www.pnas.org/cgi/doi/10.1073/pnas.1008737107Kreisel et al.
lation), they are brightly fluorescent and that they appear to ad-
cytotoxic effects have not been rigorously assessed.
Previous reports have demonstrated that the i.v. administration
of clodronate liposomes can be used to deplete blood monocytes
Q-dot–positive cells from the circulation, consistent with the flow
cytometry data that suggests that these cells are monocytes. Our
colocalize in vessels near sites of neutrophil extravasation, Q-dot–
positive cells express the monocyte surface markers CD115, F4/80,
and L6C, and finally that the clodronate-liposome-mediated de-
pletion of monocytes dramatically impairs neutrophil trans-
exclude the possibility that clodronate liposomes might deplete
other rare cell types. For example, others have described the exis-
tence of pulmonary intravascular macrophages, which—similar to
Kupffer cells in the liver—play a role in the phagocytosis of large
less prevalent in mice than in other species, they could potentially
capture clodronate liposomes from the circulation. However, it is
important to emphasize that in our transplant experiments, only
recipient mice were treated with i.v. clodronate liposomes. Based
on phagocyte depletion rates in other tissues, we would not expect
residual clodronate liposomes in the recipient animal to efficiently
deplete graft-associated macrophages in the 2–3 h window after
transplantation. Therefore, the effect of clodronate-liposome de-
pletion on neutrophil extravasation is unlikely to be due to the
depletion of tissue resident macrophages. Notably, clodronate-li-
posome treatment has been shown not to deplete or directly affect
neutrophil behavior in vitro and in vivo (30, 31).
Intravital 2P imaging showed that the depletion of blood
at the transendothelial migration step. These findings extend on
previous reports, which suggested that monocyte and neutrophil
trafficking were interdependent during pulmonary inflammation.
Inresponsetointratracheal instillationofCCL2 and LPS, alveolar
neutrophil accumulation was markedly inhibited when animals
were treated with anti-CCR2 antibody or were genetically de-
ficient in CCR2 (32). Based on experiments using bone marrow
the airspaces depended on CCR2-expressing blood monocytes
(33). Of note, this study relied on quantitative analysis of neu-
trophils within the bronchoalveolar lavage fluid and could there-
fore not provide mechanistic insight into precisely at what step
monocytes influence neutrophil recruitment. Moreover, the ex-
pression ofCCR2 onothercell types,including dendriticcells, NK
cells,mast cells, T cells and, in particular,neutrophils,complicates
the interpretation of the studies described above (34, 35). None-
theless, our results using clodronate-liposome depletion confirm
the conclusions of Maus and colleagues (33) and extend on their
findings by showing that monocytes play a specific and previously
unrecognized role in facilitating neutrophil transendothelial mi-
gration out of pulmonary vessels.
We observed dramatic neutrophil clustering in the lung after
intratracheal administration of bacteria, similar to the neutrophil
swarming behavior observed by Chtanova and colleagues in the
lymph node after s.c. infection with Toxoplasma gondii (15). In re-
sponse to infection, neutrophil clustering is not surprising because
bacteria. Moreover, others have observed focal neutrophil re-
cruitment in response to traumatic tissue damage alone, such as at
an injection site (15, 18, 19). However, ischemia reperfusion injury
inflammatory event. Therefore, our observation that neutrophils
form focal clusters in the lung after transplant was unexpected.
Analyzing neutrophil migration using a heatmap visualization
technique revealed that neutrophil chemokine gradients were typ-
icallylocal and short-lived in vivo, resembling “chemokinebombs.”
rapid secretion of preexisting chemokine stores into the vicinity.
responding to T. gondii escape from an infected cell might provide
signals that recruit late arriving neutrophils into swarms at foci of
monocytes within the majority of neutrophil clusters after trans-
plant-mediated ischemia reperfusion injury. This observation sug-
gests that monocytes might serve as a focal source of chemokines
acting on nearby neutrophils to recruit them to sites of effector
function. The dissolution of neutrophil clusters could represent
chemokine receptor desensitization, dissipation of the chemokine
gradient, or perhaps chemorepulsion. The observation that neu-
chemokine gradient. In most cases, clusters did not completely
disassociate over our imaging time window; a small number of
neutrophils typically remained in the center of a cluster. Because
neutrophilsarrestduringtheiroxidative burst, itispossible that the
nonmotile cells represent a population of activated neutrophils.
We show that 2P microscopy is a promising approach for the in
vivo study of cellular immune mechanisms that operate during
inflammation and infection in the lung. We found that the de-
pletion of blood monocytes impairs neutrophil recruitment to the
lung specifically during transendothelial migration. Time-lapse
imaging of neutrophil migration in vivo suggests that neutrophil
recruitment to effector sites and clustering is regulated by short-
range transient chemokine gradients. The association of Q-dot–
positive cells with these dynamic clusters suggests that monocytes
of neutrophils and have important implications for the design of
therapeutics to treat inflammatory lung diseases.
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 tetramethylrhod-
transendothelial migration after transplantation. Pulmonary
655-nm Q-dots. (A) Two-photon image of neutrophils (green)
extravasating from a medium-size vessel (white lines) in a
liposome (CL) treatment of the transplant recipient results in
neutrophil accumulation in medium-sized vessels and a re-
in untreated recipients (control, <5%) and in clodronate-
liposome treated recipients (CL, >90%). (Scale bar: 60 μm.)
Clodronate-liposome depletion impairs neutrophil
Kreisel et al. PNAS
| October 19, 2010
| vol. 107
| no. 42
amine (Invitrogen) (500 μg/mL) or 100,000 cfu of L. monocytogenes were ad-
ministered intratracheally after dilution in 50 μL of PBS. Clodronate liposome
suspensions were prepared as described (31). Monocytes were depleted by
(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 venti-
lator 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 GFPhighcells 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 PharMin-
gen). 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. Neu-
trophil 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
ACKNOWLEDGMENTS. We thank A. P. Gieselman for animal care. D.K. and
A.E.G. are supported by National Heart, Lung, and Blood Institute Grant
1R01HL094601 and D.K. by Grant 1K08HL083983, jointly sponsored by the
National Heart, Lung, and Blood Institute and the Thoracic Surgery
Foundation for Research and Education. M.J.M. is supported by the National
Institute of Allergy and Infectious Diseases Grant AI077600.
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