Inflammation: The first responders need help

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Editor's Introduction

Neutrophils scan for activated platelets to initiate inflammation

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White blood cells, or neutrophils, are like the EMT response to an infection. They are the first ones to arrive at an infected site, but how do these cells know where to go? How do we determine the origin of the 911 call? Neutrophils extend protrusions into blood vessels that bind to platelets in the blood. This communication between neutrophils and platelets is what helps the neutrophils determine the site of the wound. In some injuries, excessive inflammation is harmful; this paper shows that blocking neutrophil-platelet interactions in mice can reduce inflammation and, in turn, fatalities. 

Paper Details

Original title
Neutrophils scan for activated platelets to initiate inflammation
Original publication date
Vol. 346, Issue 6214, pp. 1234-1238
Issue name


Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with an asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in the redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thromboinflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and they suggest that the neutrophils' bipolarity allows the integration of signals present at both the endothelium and the circulation before inflammation proceeds.


Neutrophils are primary effectors of the immune response against invading pathogens but are also central mediators of inflammatory injury (1). Both functions rely on their remarkable ability to migrate within and through blood vessels. The migration of neutrophils is initiated by tethering and rolling on inflamed venules, a process mediated by endothelial selectins (2). Selectin- and chemokine-triggered activation of integrins then allows firm adhesion, after which leukocytes actively crawl on the endothelium before they extravasate or return to the circulation (3). A distinct feature of leukocytes recruited to inflamed vessels is the rapid shift from a symmetric morphology into a polarized form, in which intracellular proteins and receptors rapidly segregate (4). In this way, neutrophils generate a moving front or leading edge where the constant formation of lamellipodia (actin projections) guides movement, and a uropod or trailing edge where highly glycosylated receptors accumulate (56). We deemed it unlikely that this dramatic reorganization served to exclusively generate a front-to-back axis for directional movement, and we explored the possibility that neutrophil polarization functions as an additional checkpoint during inflammation.

We performed intravital microscopy (IVM) imaging of venules in cremaster muscles of mice treated with the cytokine tumor necrosis factor–α (TNF-α), an inflammatory model in which the vast majority of recruited leukocytes are neutrophils (fig. S1). Within seconds after arresting, leukocytes formed a lamellipodia-rich domain, or leading edge, and a CD62L-enriched uropod, which we could identify by its localization opposite to the leading edge and the direction of cell movement (movie S1 and Fig. 1A) (68). Confirming previous reports, we observed numerous interactions of platelets with the leading edge of adherent neutrophils [Fig. 1A and fig. S2A (810)]. During these experiments, we noticed that the uropod underwent continuous collisions with circulating platelets, a fraction of which established measurable interactions that were usually transient (Fig. 1B and movie S2). Because platelets captured by the uropod represented a substantial fraction of all interactions (31%), we searched for the receptor(s) mediating these contacts. We reasoned that PSGL-1, a glycoprotein ligand for P-selectin (11) that segregates to the uropod of polarized neutrophils (12), could be responsible for these interactions. Analysis of mice deficient in PSGL-1 (Selplg–/–mice) revealed marked reductions in platelet interactions with the uropod, whereas those at the leading edge remained unaffected (Fig. 1B). In contrast, deficiency in the β2 integrin Mac-1 (Itgam–/–) resulted in reductions at both the uropod and leading edge (Fig. 1B). In vivo labeling of Mac-1 and PSGL-1 confirmed these functional data, with Mac-1 localized throughout the cell body and PSGL-1 exclusively at the uropod (Fig. 1C). Specifically, PSGL-1 clustered in a small region of the uropod, whereas CD62L was widely distributed in this domain (Fig. 1C). Analyses of mice expressing a functional Dock2-GFP protein (GFP, green fluorescent protein), a guanine nucleotide exchange factor of Rac GTPases (13), revealed colocalization of Dock2 with PSGL-1 clusters on crawling neutrophils (fig. S3 and movie S3), suggesting active structural dynamics within this region. This observation together with the high frequency of platelet collisions with the PSGL-1 clusters suggested that this domain might be actively protruding into the vessel lumen. Using high-speed spinning-disk IVM, we could obtain three-dimensional (3D) reconstructions of polarized neutrophils within inflamed venules of Dock2-GFP mice (Fig. 1D), demonstrating that the PSGL-1 clusters indeed projected toward the vessel lumen in about 40% of adherent neutrophils, whereas in the remaining 60% of the cells, it extended laterally, parallel to the endothelial surface (Fig. 1, D and E, and movie S4). As a consequence, the luminal space of inflamed venules was populated by multiple PSGL-1–bearing clusters suitably positioned to interact with circulating cells (Fig. 1F and movie S5).


Fig. 1. Neutrophils recruited to inflamed venules interact with activated platelets via protruding PSGL-1 clusters.(A) Micrographs of polarized neutrophils interacting with platelets (red; yellow arrowheads) through the leading edge or the CD62L-labeled uropod (blue). (B) Quantification of total or domain-specific platelet interactions in wild-type mice or mice deficient in P-selectin (Selp–/–), PSGL-1 (Selplg–/–), or Mac-1 (Itgam–/–); n = 5 to 8 mice, 38 to 133 interactions. (C) In vivo receptor distribution on polarized wild-type neutrophils. (D) Examples of luminal and lateral projections from 3D reconstructions of polarized Dock2-GFP neutrophils (see also movie S4). (E) Frequency of neutrophils extending PSGL-1 clusters into the lumen (Lu), laterally (La) or between the cell body and the endothelium (En). n = 6 mice, 251 cells. (F) 3D reconstructions of an inflamed vessel showing the distribution of PSGL-1 clusters (movie S5). (G) Representative micrographs of neutrophils interacting with nonactivated (arrow) or activated P-selectin+ platelets (arrowhead), and quantification of interactions of each domain with P-selectin+ or JON/A+ platelets. n = 3 to 4 mice, 66 to 116 interactions. Scale bars, 10 μm. Bars show mean ± SEM. *< 0.05; ***< 0.001, one-way analysis of variance (ANOVA) with Tukey’s post-hoc test.
Experimental Question

The authors knew previously that on an inflammatory stimuli, the neutrophils move to the site of inflammation.

Here, they wanted to understand in detail how these cells move.
Do they need help from other cell types?
Are they interacting with any cell types?


They use intravital microscopy (IVM). This figure details the requirements to use IVM.

The following article details the technique and how it has been used in research:

Panel A

The authors stained different cells with specific colors to distinguish between the cell types. Platelets are stained red and the CD62-L ligand of neutrophils is stained blue. Because CD62-L is known to be enriched at the uropod, this staining is to indicate the uropod region.

The leading edge of the neutrophils (not stained blue) was seen to be in close proximity to the platelets, indicating that the interaction between these cell types occurs via the leading edge.

Panel B

In this, the authors quantitate information available from IVM. Because IVM can measure real-time events, they record the number of collisions between the neutrophils (WBC in the graph) with the platelets per minute. To understand the contribution of the receptors that may be involved in this interaction, they use mice that are deficient in P selectin (Selp–/–), PSGL-1 (Selplg–/–), and Mac-1 (Itgam–/–). Use of Itgam–/– mice serves as a positive control as it has been shown previously that Mac-1 plays a role in neutrophil-platelet interactions. They separate the total interactions observed into the ones observed at the leading edge and the uropods of the neutrophils.

Firstly, of the total collisions, they observe the maximum at the leading edge of the neutrophils (comparing gray bars in the three panels).

Next, within each (leading edge–uropod) interaction, they observe that only the absence of Mac-1 and not of P-selectin or PSGL-1 affects the interaction at the leading edge of the neutrophils (panel 2, comparing all the bars with the gray bar).

However, absence of PSGL-1 and Mac-1 (as well as P-selectin), does reduce the total number of interactions (panel 1, comparing with the gray bar).

Interestingly, the absence of all these receptors/ligands affects the interactions at the uropod, indicating that PSGL-1 and Mac-1 on neutrophils mediate the interactions with platelets.

Panel C

The authors look at the distribution of these different receptors on the cell surface of neutrophils. They label these receptors with different colors and use imaging to analyze where in the cell body are the receptors localized.

They observe that whereas Mac-1 (green) is widely distributed throughout the cell body, CD62-L and PSGL-1 are localized only in the uropod region.

Within the uropod region, they observe that whereas CD62-L is widely distributed, PSGL-1 seems to cluster together in a very small region.

These observations also help in understanding what the authors observed in Figure 1B.

Panel D

This is a representation of the movie the authors were able to obtain on 3D reconstructions of the IVM images.

Panel E

This is a graphical representation of data obtained in Figure 1D.

Approximately 40% of the neutrophils projected their PSGL-1 clusters into the lumen of vessels, whereas 60% of them projected it laterally.

Panel G

Previous observations were suggestive that not all platelets were interacting with neutrophils at the uropod. The authors thus wanted to find whether there was a subset of platelets that were able to do this. For this, they labeled P-selectin in vivo (in mice).

They observed that all the P-selectin+ platelets interacted with the neutrophils at the uropod, whereas interactions at the leading edge did not necessarily depend on P-selectin expression.

The observation that only a small fraction of circulating platelets engaged in interactions with the uropod prompted us to search for subsets of platelets prone to this behavior. In vivo labeling for P-selectin or for active β3 integrins revealed that virtually all platelets interacting with the uropod were activated (P-selectin+ or with active β3 integrins), whereas a fraction of those engaging the leading edge were not (Fig. 1G and figs. S2B and S4). These findings further suggested that P-selectin present on the surface of activated platelets might be mediating the interactions with the PSGL-1 clusters. Analyses of mice deficient in P-selectin (Selp–/– mice) indeed demonstrated patterns of platelet interactions with the two leukocyte subdomains that were similar to those found in mice lacking PSGL-1 (Fig. 1B). These results indicated that neutrophils recruited to inflamed vessels extend a PSGL-1–bearing microdomain into the vessel lumen that scans for activated platelets present in the bloodstream through P-selectin.

During the course of our IVM experiments, we also noticed alterations in the intravascular behavior of adherent neutrophils in the different mutant mice. Deficiency in Mac-1 severely compromised neutrophil crawling on the inflamed vasculature (Fig. 2, A and B), a process previously reported to be mediated by this integrin (3). Surprisingly, although PSGL-1 was excluded from the area of contact with the endothelium (Fig. 1, D and E), neutrophils deficient in this glycoprotein also displayed reductions in crawling displacement and velocity (Fig. 2, A and B), and these defects were cell-intrinsic (fig. S5 and movie S6). To exclude potential defects originating from PSGL-1 contributions in the early steps of leukocyte recruitment by binding endothelial P-selectin (14), we prevented PSGL-1 binding to P-selectin using a blocking antibody injected after neutrophils had adhered. Inhibition at this stage did not affect leukocyte adhesion to inflamed venules but specifically decreased interactions with the uropod (fig. S6) and caused reductions in crawling kinetics (Fig. 2, A and B). We thus speculated that the engagement of PSGL-1 at the uropod might promote crawling of polarized neutrophils. To test this hypothesis, we first induced transient depletion of platelets, a treatment that resulted in virtual suppression of crawling (Fig. 2, A and B, and fig. S7A). We next analyzed two models in which neutrophil polarization or signaling through PSGL-1 was impaired. In the first model, we induced hematopoietic-specific deletion of the gene encoding Cdc42 (fig. S8A), a small Rho-GTPase required for neutrophil polarization (15). Confirming previous in vitro observations, Cdc42-deficient neutrophils were unable to form a leading edge–to–uropod axis and instead formed multiple protrusions, lacked a distinguishable uropod, and failed to form PSGL-1 clusters in vivo (fig. S8B). Impaired polarization in these mutants compromised interactions between neutrophils and circulating platelets (fig. S8C), and neutrophils in these mice displayed severely impaired crawling kinetics (Fig. 2, C and D). In the second model, we analyzed mice in which PSGL-1 is normally distributed at the cell surface and can interact with P-selectin but cannot propagate outside-in signals because of the absence of the cytoplasmic domain [PSGL-1ΔCyt mice (16)]. Although neutrophil adhesion to TNF-α–stimulated vessels was partially compromised in PSGL-1ΔCyt mice because of reductions in the surface levels of PSGL-1, those cells that adhered polarized normally (fig. S9A) and displayed marked reductions in crawling kinetics (Fig. 2, C and D) despite elevated levels of Mac-1 on the surface (fig. S9B). Thus, polarization of a signaling-competent PSGL-1 drives the intravascular migration of neutrophils.


Fig. 2. PSGL-1 controls intravascular motility and the distribution of Mac-1 and CXCR2. (A) Tracks of crawling neutrophils within inflamed vessels in untreated wild-type mice, mice deficient in PSGL-1 (Selplg–/–) or Mac-1 (Itgam–/–), and mice depleted of platelets by antiplatelet serum or treated with a PSGL-1–blocking antibody. (B) Quantification of the crawling displacements and instantaneous velocities of neutrophils in the same groups as (A); n = 50 to 56 cells, 4 to 9 mice. (C) Tracks of neutrophils with conditional deletion of Cdc42 or expressing a mutant form of PSGL-1 that lacks the cytoplasmic tail (PSGL-1ΔCyt) and (D) quantification of the displacement per minute and instantaneous velocities of adhered neutrophils. n = 50 to 55 cells, 3 to 5 mice. (E) Representative micrographs and quantification (F) of the in vivo distribution of CXCR2 and Mac-1 in polarized neutrophils from wild-type and PSGL-1–deficient mice; n = 17 to 19 cells, 3 mice. Scale bar, 10 μm. Data show mean ± SEM. *< 0.05; ** P < 0.01; ***< 0.001; ANOVA with Tukey’s multigroup test (B) or unpaired t test (F).
Panel A


The authors wanted to use mathematical tools to determine the displacement of neutrophils in the blood vessels. They transported images obtained from IVM and used the X and Y axis coordinates of the cells to calculate the movement of each cell.

Experimental setup:

To understand the role of PSGL-1 in mediating neutrophil movement, the authors compared the displacement in WT neutrophils with that from Selplg–/–, Itgam–/– mice. They also used pharamacological blocking of PSGL-1 and depleted platelets to study the effect of neutrophil displacement in these conditions.


In neutrophils from mice where platelets were depleted, PSGL-1 was blocked or with deficiency in Mac-1 showed impairment in neutrophil mobility. There was a slight reduction in mobility in neutrophils from mice that lacked PSGL-1.

To read more on how to calculate displacement of cells:

Panel B

The authors quantitated what was observed from Figure 2A.

Mice that lacked PSGL-1 and Mac-1 showed decreased neutrophil crawling as measured by velocity and displacement. Mice where platelets were depleted and with pharmacological blocking of PSGL-1 also exhibited similar reduction in neutrophil crawling.

Panel C

Similar experimental setup as observed in Figure 2A. In this experiment they generated conditional knockout mice.

Cdc42 is required for neutrophil polarization and the authors have observed that mice deficient in this molecule are unable to form PSGL-1 clusters.

PSGL-1/cyt: These mice lack the cytoplasmic tail of PSGL-1 and hence, although PSGL-1 is distributed normally, the signal induced cannot be transmitted through the cell.

As observed with deletion and blocking of PSGL-1, in these conditional knockout mice also, the authors observe a significant reduction in neutrophil crawling.The authors performed several variations of the experiments to confirm the role of PSGL-1 in neutrophil-platelet interaction.

Panel D

Quantification of figure 2C.

Panels E and F

In these, the authors wanted to understand the pathway that was involved in PSGL-1 mediated neutrophil crawling. Since Mac-1 and CXCR2 are two widely accepted receptors that help neutrophils tmigrate through blood vessels, the authors looked at the distribution of these two receptors on the cell surface and quantitated their expression at the leading edge and uropod.

The authors observed a homogenous expression of Mac-1 on WT neutrophils, however CXCR-2 clustered on the leading edge. The use of staining for CD62L in this helps distinguish the leading edge from the uropod. Loss of PSGL-1 altered the distribution of these two receptors.

To search for possible mechanisms by which PSGL-1–derived signals promoted crawling, we analyzed the in vivo distribution of Mac-1 and the chemokine receptor CXCR2, two receptors required for the intravascular migration of neutrophils (317). In wild-type cells, Mac-1 was homogeneously distributed throughout the cell body, whereas CXCR2 preferentially localized at the leading edge (Fig. 2E and movie S7). Neutrophils deficient in PSGL-1 exhibited a mislocalization of both receptors (Fig. 2E, fig. S10, and movie S8). These alterations were even more dramatic in wild-type mice upon platelet depletion (figs. S7B and S10 and movie S9), which agreed with the suppression of crawling in these mice (Fig. 2A). The absence or inhibition of PSGL-1 in Mac-1–deficient mice did not lead to further reductions in platelet interactions or crawling kinetics (fig. S11), indicating that these receptors function along the same pathway and that additional platelet-derived mediators and unknown neutrophil receptors that mediate platelet interactions can regulate crawling. Thus, intact distribution of and signaling through PSGL-1 at the uropod regulates neutrophil crawling, at least in part by orchestrating the appropriate distribution of adhesive and chemotactic receptors.

We next explored how this phenomenon might contribute to pathogenic inflammation. We used a model of acute lung injury (ALI) in Balb/c mice that closely simulates transfusion-related ALI (18). In this model, the transient elimination of neutrophils or platelets protects from death [Fig. 3A and (19)], indicating that this might be an appropriate model to study the functional partnership between these cells. Intravital analyses of the cremaster microvessels in ALI-induced mice confirmed the findings in crawling kinetics, receptor distribution, and luminal or lateral projections made using TNF-α (fig. S12 and movie S10) and further revealed that during ALI, the uropod becomes the predominant domain for platelet interactions, which contrasted with the preferred use of the leading edge in the nonpathogenic TNF-α–induced model (Fig. 3, B and C). Interactions at the uropod during ALI were mediated by PSGL-1, whereas Mac-1 mediated interactions with both domains (Fig. 3C and fig. S13). We obtained similar responses in a model of endotoxemia (Fig. 3D), indicating that during pathological inflammation, the uropod becomes the dominant interacting domain for circulating platelets.


Fig. 3. PSGL-1 at the uropod becomes a preferred docking site for platelets during pathological inflammation.(A) Survival curves of Balb/c mice treated with lipopolysaccharide (LPS) alone or LPS plus anti–MHC-I (MHC, major histocompatibility complex) to induce ALI. Neutrophils were depleted using anti-Ly6G, and platelets using antiplatelet serum before the induction of ALI; n = 5 to 20 mice. (B) (Left) Representative micrographs of inflamed venules during ALI. The asterisks indicate platelets interacting with the uropod of neutrophils. (Right) Quantification of platelet interactions with the leading edge or uropod in control (LPS only) and ALI-induced mice. Scale bar, 10 μm. n = 3 to 4 mice, 32 to 73 interactions. (C) Frequency of interactions with the leading edge or uropod in TNF-α–treated or ALI-induced mice, and distribution of interactions in wild-type mice and mice deficient in PSGL-1 (Selplg–/–) or Mac-1 (Itgam–/–); n = 3 to 5 mice, 23 to 137 interactions. (D) Frequency of interactions with the leading edge or uropod during sepsis in wild-type mice and mice deficient in PSGL-1 or Mac-1. n = 3 to 4 mice, 32 to 56 interactions. Bars show mean ± SEM. *< 0.05; ***< 0.001 as determined by ANOVA with Tukey’s multigroup test.
Figure 3

The authors test their findings of neutrophil-platelet interaction during infection/injury–related inflammatory conditions.

Panel A

Here the authors wanted to determine the role of neutrophil mortality because of acute lung injury. ALI was induced by injecting mice with LPS (an endotoxin that can cause septic shock) followed by anti–MHC-I antibody.

This closely mimics transfusion-related ALI. Platelets or neutrophils were depleted prior to ALI induction in some groups of mice.

Whereas LPS alone treated mice survived though the experimental study, mice where ALI was induced showed increased mortality. However, depleting neutrophils or platelets reduced the susceptibility of mice to ALI-related mortality.

Panel B

In this the authors examine the interactions between platelets and neutrophils in inflamed blood vessels through IVM.

Similar to Figure 1, the CD41 (red) labeled platelets are seen to be interacting with the CD62L-enriched uropod of neutrophils during ALI. The bar graph on the right side is the quantification of the interactions observed by IVM. During ALI, the number of interactions between the neutrophils and platelets is significantly increased, specifically at the uropod region.

Panels C and D

The authors quantitate from IVM the number of interactions/collisions in different genotypes of mice during different conditions.

3C first panel:

Here, they observed the number of interactions between WT cells at the leading edge and the uropod that appears when mice are treated with TNF-a.

3C second panel:

Here, they induce ALI in WT, Selplg–/–, and Itgam–/– mice. Whereas the number of interactions increases at the uropod in WT mice, this is dependent on PSGL-1 and Mac-1. In the absence of these two receptors, the number of interactions between neutrophils and platelets is significantly reduced at the uropod.


The authors examine whether PSGL-1 and Mac-1 are required for interactions during sepsis. Again, loss of these two receptors reduces the number of interactions at the uropod.

To test whether the engagement of PSGL-1 at the uropod was causally related to neutrophil-mediated vascular inflammation, we explored its contribution in the model of ALI. Intravital imaging of the pulmonary microcirculation revealed a rapid increase in platelets captured by recruited neutrophils that were strongly inhibited by blocking PSGL-1 (fig. S14). In addition, deficiency in PSGL-1 or Mac-1, or inhibition of PSGL-1, resulted in moderate protection from ALI-induced death (Fig. 4A and fig. S15A). The use of computed tomography to track pulmonary edema over time revealed partial protection from ALI in mice deficient in Mac-1 and almost complete protection when PSGL-1 interactions were blocked (Fig. 4, B and C). This protection correlated with reduced neutrophil infiltrates in the lung (fig. S16) and suggested that interactions at the uropod critically contribute to vascular injury. Deficiency in either receptor or inhibition of PSGL-1 also prevented hepatic damage during endotoxemia (Fig. 4D and fig. S15B). Consistent with previous reports (2021), we detected elevations in the plasma levels of neutrophil-derived extracellular traps (NETs) during ALI and sepsis. These elevations were completely blunted when platelets were depleted, by blocking PSGL-1, or in the absence of Mac-1 (fig. S17), suggesting that other forms of neutrophil activation can be triggered upon platelet interactions through PSGL-1.


Fig. 4. PSGL-1–mediated interactions trigger vascular injury.(A) Survival curves of Balb/c mice treated with LPS alone or LPS plus anti–MHC-I to induce ALI. The absence of Mac-1 or inhibition of PSGL-1 protects from death; n = 5 to 19 mice. (B) Representative axial slices of the thorax of Balb/c mice at different times after induction of ALI. The white signal in the pulmonary space identifies edema, which is quantified in (C); n = 7 to 8 mice per group. (D) Quantification of hepatic injury as levels of AST and ALT transaminases in plasma of the indicated group of mice 24 hours after treatment with LPS; n = 7 to 11 mice. (E) Representative brain sections of wild-type mice 24 hours after inducing ischemia, showing vessels at increasing magnifications and intravascular neutrophil (Ly6G, green)–platelet (CD41, red) aggregates. Scale bars, 10 μm. (F) Percentages of infarcted hemispheres 24 hours after arterial occlusion in control wild-type mice, mice deficient in Mac-1 (Itgam–/–), and wild-type mice after blocking PSGL-1. Images are representative brain sections stained with TTC, showing the extent of ischemia as white areas with a red outline; n = 5 to 8 mice. Bars show mean ± SEM. *< 0.05; **< 0.01; ***P< 0.001, ANOVA with Tukey’s multigroup test.

Figure 3 summarized experiments conducted to show that neutrophils, platelets, and their interaction were all critical components of the inflammation response and therefore were contributing to mouse susceptibility to ALI-related death.

Figure 4 will now provide evidence supporting the authors' claim that PSGL-1 is the ligand responsible for the interaction between the neutrophils and platelets. This will also identify if the PSGL-1 activity at the tail end of the neutrophil cell is specifically contributing to blood vessel inflammation as a result of an injury.

Panel A questions

This panel is similar to Panel A in Figure 3.

1) What is the main difference between Panel A in Fig. 3 and Panel A in Fig. 4?

2) The panel depicts survival curves. Each individual mouse can only survive (or not survive) one time; so, how are the authors able to plot survival over a period of time?

Panel A answers

1) Both graphs depict survival curves; in other words, the % survival over a specific period of time. Panel A in Figure 3 examines how mice with the same genetic background respond to different treatments that aren't specifically examining the role of PSGL-1. Panel A in Figure 4 compares the survival of mice subjected to different treatments or genetic backgrounds explicitly related to PSGL-1; specifically, it compares a control group (LPS) to different groups of mice all treated to induce ALI. The WT mice have both PSGL-1 and Mac-1 functioning normally; Itgam-/- mice don't have functioning Mac-1 cell adhesion molecules; and PSGL-1-blocked mice have no functioning PSGL-1 ligands.

2) Survival curves examine the average survival of a population, or group, over a period of time. In this panel, each line represents the % of surviving mice (# mice alive/total # of mice in that group) within each treatment group of mice. The graph shows that mice treated with only LPS survived the trial. Wildtype (wt) mice treated to induce API had the lowest survival rate. Mice without functioning Mac-1 ligands that were treated to induce API had a greater chance of survival than the wildtype mice, but the mice with the best chance of surviving induced API were those with no functioning PSGL-1.

By inhibiting PSGL-1, the authors observed that mice were protected from ALI-induced death because the interaction between neutrophils and platelets was inhibited.

Panels B and C

The authors now begin to examine a variety of different tissues to verify that all neutrophil-mediated inflammation is regulated by PSGL-1. Panels B and C specifically look at differences in accumulation of fluid in the lungs (also called pulmonary edema). When PSGL-1 is blocked, accumulation of fluid during induced ALI was reduced.

Panel B shows images from computerized tomography (CT) scans, a form of X-ray imaging. The white spaces indicate a build-up of fluid.

Panel C displays a quantification of the amount of fluid building up (in this case, the % of white space visible in CT scans taken over time) for each treatment group.

Panel D

For this experiment, the authors induced endotoxemia, which can result in septic shock, by injecting mice with LDS from E. coli bacteria. This graph depicts liver damage in the different treatment groups, measuring two different liver enzymes (AST and ALT). Increased levels of both enzymes are found during "ischemic hepatopathy," also known as shock liver, which is something expected during endotoxemia.

Panels E and F

Finally, the authors move on to examining damage in brain tissue.

The authors induced a type of stroke, focal brain ischemia, via a method that causes a blood clot to block the middle cerebral artery.

Panel E shows adhesion of neutrophils (in green) to platelets (in red) within the blood vessels in the brain as a response to the injury.

Panel F compares area of "infarction", or reduced/halted blood supply, to the brain for different groups of mice. When PSGL-1 is blocked, the infarction resulting from the induced stroke is greatly reduced.

Finally, we examined whether PSGL-1–mediated interactions also underlie ischemic injury, a prevalent form of vascular disease (22). We used a model of stroke triggered by permanent occlusion of the middle cerebral artery, in which neutrophil depletion significantly reduces tissue death as measured by the percentage of infarcted hemisphere [fig. S18 and (23))] Interactions between neutrophils and platelets within the microvasculature of infarcted brains were inhibited by blocking PSGL-1 (Fig. 4E and fig. S19), and this correlated with significant reductions in infarct volumes when PSGL-1 was inhibited or in the absence of Mac-1 (Fig. 4F).

We have uncovered a critical checkpoint during the early stages of inflammation: Neutrophils recruited to injured vessels extend a domain into the lumen, where PSGL-1 clusters scan for the presence of activated platelets. Only when productive interactions occur do neutrophils organize additional receptors needed for intravascular migration or generate NETs, and inflammation ensues (fig. S20 and movie S11). Our findings reveal that the dynamic reorganization of neutrophil domains and receptors allows simultaneous interactions with both the vascular wall and activated platelets in the circulation to provide a rapid and efficient regulatory mechanism early during inflammation.

Supplementary Materials

Materials and Methods
Figs. S1 to S20
Tables S1 and S2
References (2429)
Movies S1 to S11

References and Notes

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