It has been argued that the mechanical mechanism of cell squeezing could stimulate their margination promoting interaction with the lung capillaries, an interesting hypothesis that requires further evidence Kuebler and Goetz, ; Rossaint and Zarbock, We have recently directly shown, using IVM to image the pulmonary capillary network of mice that a single dose of the CXCR4 antagonist, AMD, did not compromise the lung intravascular retained pool of neutrophils under homeostatic conditions Pillay et al.
Further applying dynamic planar gamma scintigraphy, we have also shown that AMD does not affect the retention of primed neutrophils in the capillary circulation of the lung in humans. Taken together these data suggest that the CXCR4-CXCL12 chemokine axis does not support neutrophil retention in the lung microvascular in either mouse or human Pillay et al.
Thus to date the precise molecular mechanisms underlying the retention of mature neutrophils in the pulmonary capillaries are unknown or still remain a mystery. Figure 2.
Spatial organization of neutrophils in the lung and spleen during homeostasis. A Frame from lung IVM video showing marginated neutrophils red within the microvessels of the lung green.
B Precision cut spleen slice showing tissue resident neutrophils red and margiant zone macrophages green delimiting the MZ. While neutrophils serve as a critical line of host defense in the lung, in the context of ARDS and a number of chronic lung diseases, e.
Thus, understanding the mechanisms underlying neutrophil influx is desirable to enable the development of targeted therapeutics that can reduce neutrophil numbers in these clinical scenarios. In contrast to the situation under homeostasis, there is consistent evidence from several studies showing the requirement of specific adhesion molecules to support increased neutrophil retention within the microvessels of the lung and their further migration within the infected parenchyma Doerschuk, L-IVM showed that following systemic challenge, E.
This work indicates that the lung is an important host defense niche for the detection and capture of systemic pathogens, and requires cooperation between the vascular endothelium and marginated neutrophils Yipp et al. In the search for new adhesion molecules that support neutrophil retention in the lung during inflammation, an in vivo functional screen surprisingly identified dipeptidase-1 DEPEP1 Rajotte and Ruoslahti, ; Choudhury et al. DEPEP1, a membrane enzyme expressed by activated pulmonary endothelium, was shown to support neutrophil adhesion, independent of its enzymatic activity.
Moreover, genetic deletion studies and use of a blocking peptide showed that neutrophil adhesion and recruitment in the inflamed lung was significantly attenuated in the absence of DEPEP1 during sepsis Choudhury et al. Finally, when the DEPEP1 blocking peptide was used therapeutically in mice administered with a lethal dose of LPS, it showed a remarkable survival effect and an impressive reduction in neutrophil recruitment into the inflamed lung Choudhury et al.
With respect to the focus of this review, highlighting how IVM has been key to advancing our understanding and identifying the molecular pathways regulating neutrophil trafficking, this research is notable in that the initial screen involved using confocal IVM to identify a peptide that localized to both the lung and liver endothelium after LPS treatment and reduced neutrophil accumulation in these tissues.
Importantly, while this research was carried out in mice, recombinant human DPEP1 supported the adhesion of human neutrophils in vitro , indicating its translational potential. While neutrophils are important for host defense, as mentioned above, when they accumulate in large numbers in tissues, they also have the potential to cause considerable damage to the host tissue.
This is due to the fact that their granule proteins and neutrophil extracellular traps NETs , both important for their anti-microbial functions, are also cytotoxic. In this respect, a series of recent discoveries, also made using IVM, provide an explanation for why marginated neutrophils in the lung may not be as cytotoxic as those in the circulation. Following their release from the BM neutrophils only circulate for 6—10 h before they exit into tissues, including the spleen, BM, and lung Casanova-Acebes et al.
At this time, as compared to neutrophils freshly mobilized from the BM, circulating neutrophils exhibited changes in their proteome, with a reduction in cytotoxic granule proteins, which in turn reduced their ability to form NETs. They also explain why acute lung injury caused by the influx of neutrophils in response to an inflammatory stimulus varies considerably dependent on the time of day.
Thus in mice, LPS challenge of the lungs at night will result in greater host tissue damage, due to accumulation of neutrophils from the circulation that have a high content of cytotoxic proteins in their granules as compared to those that would accumulate during the day that have an aged phenotype with lower cytotoxic potential Adrover et al. While these studies have been performed in mice, similar changes in neutrophil proteome have been reported human neutrophils with aging, suggesting that these findings are translatable Adrover et al.
Another fascinating function of neutrophils has been identified by Wang et al. IVM showed that following laser injury of the liver, tissue neutrophils were involved both in dismantling the injured vessels and then in directly contributing to the deposition of collagen in a honeycomb pattern to create a path to rebuild the new vasculature Wang et al.
In response to the hepatic injury, some neutrophils migrated away from the site of injury while a small number were observed to re-enter the circulation.
While it is fascinating that a subset of neutrophils, having experienced tissue injury in one organ, makes a pit-stop in the lungs before being cleared in the BM, the reason for this process is still not fully understood.
More models need to be tested in order to prove whether this is a specific mechanism that links the liver and the lung or broadly applies to any injured organ, and whether this is only linked to sterile damage or applies also to infection.
Recently, Fluorescent influenza virus color-flu has been developed as a means of studying influenza infection in the lungs of mice by IVM. Details of the model and a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with Color-flu for multicolor analysis have also been reported Xie et al. Using this model, pulmonary permeability by dextran leakage from the lung vessels into the alveolar space and blood flow speed by i.
Thus they reported a reduction in pulmonary permeability and blood perfusion speed during infection, highlighting the severe pulmonary damage created by the virus to the host Ueki et al.
As a secondary lymphoid organ, an important function of the spleen is in mounting the adaptive immune response during pathogen challenge. In addition, three distinct subsets of macrophages metallophilic, marginal, and red pulp present in the spleen play a critical role in filtering the blood, removing senescent red blood cells and systemic pathogens.
After the BM the spleen contains the largest number of neutrophils during homeostasis, but until recently the dynamics of these tissue neutrophils was unknown Casanova-Acebes et al. IVM showed that two distinct splenic neutrophil populations, at distinct maturation stages, populate the red pulp of this organ under homeostasis Figure 2B and differentially respond to pathogen challenge Deniset et al.
Their migratory speed declined 24 h after challenge increasing the dwelling time and number of firm interactions with local splenic macrophages. Ly6G int ermediate are immature and static neutrophils capable of undergoing emergency proliferation during pathogen challenge contributing to the removal of pathogen and of plucking S. The very same preNeu, immature Neu, and mature Neu that populate the BM described in detail above were also found in the spleen under homeostasis even if in a reduced number compared with the population in the BM Evrard et al.
During sepsis, preNeu numbers in both the BM and spleen expanded with a greater fold of increase in the spleen indicating that both organs contribute to emergency granulopoiesis in response to infections Evrard et al. The possibility that neutrophils can complete the last stages of their maturation outside of the BM in the spleen also give rise to the possibility that tissue specific education may prime neutrophils such that they are better tailored to the immune surveillance property of the spleen for a fast and more effective response to pathogens or tissue damage.
Puga et al. It is still unknown whether these two populations identified in humans are comparable to Ly6G high and Ly6G i n t e r m e d i a t e murine neutrophils identified by Deniset et al. The molecular mechanisms underlying the retention of splenic neutrophils are still under investigation.
Applying IVM, Pillay et al. These data suggest that the spleen can also functions as a sink, lowering the number of circulating neutrophils when they reach a specific threshold. It is possible that the pooling of neutrophils in the spleen protects other more fragile organs, such as the lung from neutrophil overload and potential damage. Intravital microscopy also revealed that neutrophils patrol unstimulated draining lymph nodes of the skin, lung, and gastrointestinal track Lok et al.
They represent a phenotypically distinct subset of neutrophils when compared with circulating neutrophils with a high level of major histocompatibility complex II MHCII high with the potential of influencing the adaptive immune system Lok et al. In contrast to other organs, neutrophil entry into the lymph nodes did not follow circadian rhythm. These temporarily resident neutrophils survey the tissue for pathogens and following bacterial infection, recruit additional neutrophils but not after sterile injury suggesting that lymph node neutrophils are able to discriminate the nature of the insult and respond accordingly Bogoslowski et al.
Intravital microscopy has been pivotal in documenting the dynamic influx of neutrophil from inflamed tissues into the lymph nodes in response to infection Hampton et al. Circulating and tissue-resident neutrophils have been shown to use both the afferent lymphatics of the infected tissue as well as HEV to enter the lymph nodes Chtanova et al.
They seem to use different molecular mechanism to enter the lymph nodes; CCR7 is essential for neutrophils to enter via afferent lymphatics Beauvillain et al. The molecular mechanisms of neutrophil entry and their physiological and pathological implications have been reviewed by Voisin and Nourshargh Intravital microscopy showed the entry of neutrophils into the popliteal lymph node PLN via multiple hotspots on HEV following influenza vaccination Pizzagalli et al.
Moreover, neutrophil positive for influenza virus were tracked entering into the PLN after vaccination and showed over a time of 2 h changes in their dynamic motility with a decrease in instantaneous and mean speed, directionality, displacement, and an increase in the arrest coefficient suggesting an increase in cell-to-cell interactions Pizzagalli et al. Five distinct neutrophil migratory behaviors have been observed: flowing, arrested, patrolling, directed migration, and swarming Pizzagalli et al.
During swarming, neutrophils were observed forming clusters that enlarged over time in areas rich in tissue resident macrophages Pizzagalli et al.
In a model of S. Moreover, by imaging lymphocytes, neutrophils and fluorescently labeled pathogens at the same time, Kamenyeva et al. Moreover, when four consecutive neutrophils enter a capillary with branches, two alternative patterns were observed, left-right-left-right or vice versa.
This pattern has been explained by the fact that when a neutrophil is traveling along a capillary of the lymph node, it reduces the chemoattractant gradient in the capillary segment where it has just traveled in and increases the hydraulic resistance of the capillary it is occupying, hence the following neutrophil uses the opposite branch to continue its journey where the chemokine gradient is higher and hydraulic resistance lower Wang et al.
Two-photon scanning-laser microscopy has provided information on the coordinated migration pattern of neutrophils within the draining lymph nodes after tissue infection showing that neutrophils can swarm and form small, large, transient, or persistent clusters within the lymph nodes Chtanova et al. Visual imaging over time revealed that neutrophils show a direct migration rather than random within the lymph node with a high average speed of Moreover, visual imaging helped in defining that neutrophil swarming is initiated by pioneering neutrophils that come together within the first minutes followed by a massive influx of neutrophils later on Chtanova et al.
These data show that not all the neutrophils that infiltrate infected areas die in situ. At least some can re-enter either the blood vessels or the lymphatics and localize within the draining lymph nodes. These exciting studies show that neutrophils have many more functions beyond the direct killing of pathogens and tissue repair, including the recruitment and activation of other leukocytes, modulation of the adaptive immune system, antigen presentation, and blocking pathogen dissemination beyond the lymph nodes.
In the aging process of a cell, senescence represents a step before apoptosis. Senescent neutrophils characterized by an increase in cell surface level of CXCR4 selectively return to the BM at the end of their life for clearance Martin et al. This mechanism of clearance represents a homeostatic signal that modulates hematopoietic niches in the BM and that regulates appearance of progenitors into the circulation Casanova-Acebes et al.
Under homeostasis, spontaneous clearance of CD62L low neutrophils from the circulation follows a circadian rhythm with an accumulation during the light time between zeitgeber times ZT ZT5-ZT13 and clearance from the circulation during the night time ZTZT5 in mice Casanova-Acebes et al. While many studies have successfully imaged the BM via IVM, capturing the migration of neutrophils across the BM sinusoidal endothelium as they are mobilized into the blood, the uptake of senescent neutrophils by macrophages has proven extremely challenging, as well as quantitative analysis of these processes Adrover et al.
Interestingly, the molecular profile of senescent neutrophils CD62L low and CD11b high resembles one of the activated neutrophils. As suggested by Casanova-Acebes et al. In fact, there are several studies showing that compromised clearance of cells leads to an unbalanced homeostasis and loss in vascular protection Adrover et al.
Further CXCR2 has been shown to promote aging during the day while CXCR4 prevents it, suggesting that both chemokine receptors are responsible for controlling the process of neutrophil aging Adrover et al.
Bone marrow is not the only organ where aged neutrophils can be cleared. This function is equally shared between the BM, spleen and liver Suratt et al. However, the molecular mechanism of senescent neutrophil clearance in the spleen and liver is not fully understood Furze and Rankin, b. IVM imaging of the spleen treated with AMD showed an increased number of splenic neutrophils but not activation or changes in CXCR4 expression suggesting that the process of senescent neutrophil clearance in the spleen is CXCR4 independent Pillay et al.
The dynamics and molecular mechanisms underlying neutrophil trafficking in homeostasis and disease have been extensively studied by IVM over the last decades, with technological advances allowing researchers to continually uncover new functions and facets of these fascinating cells Figure 3.
Thus while the original seminal studies led to the generation of the adhesion cascade paradigm tethering rolling, adhesion transmigration increasingly more sophisticated IVM, together with availability of fluorescent reporter mice, and genetically modified mice has revealed increasing levels of complexity to this process Girbl et al.
Moreover, technological advances that have allowed imaging of tissues including the lung, spleen, and lymph nodes have led to an understanding that neutrophil responses are both tissue and pathogen specific, but moreover that there are distinct subsets of neutrophils in these tissues that have different responses and functions.
Finally, the temporal nature of neutrophil responses has been revealed by IVM, whether that is early versus late response to a pathogen, or differential responses dependent on the time of day or night. This ever increasing level of complexity means that we are now in a stronger position to understand neutrophil related diseases and design targeted therapies.
Going forward IVM is a technique that could help to test in vivo the mechanism of action of drugs and help design more potent or specific therapeutics, as has been shown recently with the identification of DEPEP1 the adhesion molecule for neutrophil retention in the lungs following LPS challenge, that could be a potential therapeutic target for ARDS Choudhury et al.
In addition to having more knowledge to specifically ameliorate inflammatory pathologies we can now start to understand in more detail, the function of tissue resident neutrophils, the different subsets of these cells, and the choreography of their recruitment, tissue retention, and maturation thanks to IVM.
Figure 3. Mice are deeply anesthetized, fluorescent Abs are intravenously injected, and internal organs are partially exposed to be imaged by IVM while maintaining blood flow. Dynamic behaviors of tissue neutrophil motility, cell—cell interactions, and temporal change in speed are recorded over time. KD performed the literature search and wrote the manuscript. SR contributed to the writing and revision of the manuscript.
Both authors contributed to the article and approved the submitted version. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The slightly altered cholesterol level of the harvested neutrophils in stored blood when compared to fresh blood partially explains some of the detected differences. Keywords: antimicrobial activity; life span; neutrophil extracellular trap NET formation; neutrophils; phagocytosis; porcine blood; reactive oxygen species ROS.
Intracellularly, there are proteins recruiting phosphotyrosine phosphatases, which deactivate receptors on the surface. For example suppressor of cytokine signaling 3 down regulates G-CSF receptor signaling by blocking the phosphotyrosine on the activated receptor thereby preventing the interaction with STAT3.
Extracellularly, neutrophils and macrophages partner in the termination of inflammation[ 71 ]. The receptor Chem R23 on macrophages, DCs and endothelial cells mediates activation of macrophages that enhances the phagocytic capacity of macrophages for uptake of apoptotic neutrophils. Neutrophil apoptosis itself is a specific process with different signals triggering apoptosis via different pathways. This process is described in detail elsewhere[ 73 , 74 ]. Importantly, phagocytosis by macrophages reduces the risk of necrotic neutrophil death and down regulates the local G-CSF production to limit neutrophil activation[ 1 ].
Other deactivating processes are granule-proteins like LL and cathepsin G that stimulate rolling monocytes to migrate into the inflamed tissue. Neutrophil-derived proteins then stimulate the extravasated monocytes to maturate into macrophages and subsequently phagocytose apoptotic neutrophils. The macrophages in turn release anti-inflammatory mediators such as IL, further limiting the damage neutrophils do to host tissues[ 71 ]. Importance of these deactivating signals is seen in clinical settings such as cystic fibrosis, in which neutrophils are insensitive to signals as IL and corticoids[ 75 , 76 ].
For a long time, neutrophils were thought to only be recruited to the inflamed tissue, act as phagocytic cells, release lytic enzymes and produce ROS, after which they were cleared. However, additional functions of neutrophils in inflammatory sites have recently been described. First of all, neutrophils were shown to express genes encoding inflammatory mediators[ 2 ]. Secondly, neutrophils were found to produce anti-inflammatory molecules and factors promoting the resolution of inflammation, as described above and elsewhere[ 56 , 77 ] and thirdly, neutrophils were shown to engage in interactions with different cells of the immune system[ 20 ].
These new insights are very important for our understanding of inflammatory diseases, their resolution and possibility of neutrophils as targets to modulate immunity.
In vitro interactions with neutrophils have been shown for monocytes[ 78 ], macrophages, DCs, natural killer NK cells, lymphocytes and mesenchymal stem cells in the tissues and were reviewed by[ 79 ] Figure 2. Also, crosstalk with platelets[ 80 ] and regulatory T cells are described[ 81 ]. Subsequently, mature DCs induce T cell proliferation and polarization towards a Th1 response. Deactivated DCs showed a reduced phagocytic activity, thereby preventing the phagocytosis of neutrophils.
Second, an interaction was unraveled between neutrophils and NK cells. Neutrophils are required both in the bone marrow as well as in the peripherial development of NK cells[ 83 ]. NK cells can in turn promote neutrophil survival, expression of activation markers, priming of ROS production and cytokine synthesis[ 84 ]. These effects have only been described for neutrophils in vitro , so further in vivo investigation is needed, but it gives new insights in the expanding functions of inflammatory site neutrophils.
The importance of these additional functions is still elusive. A third interaction is reported for neutrophils and lymphocytes. Neutrophils also play an important role in B-cell help where they can even induce class switching of B-cells, a property solely assigned to T-cells[ 20 ].
The next interaction described is the crosstalk with platelets. In transfusion-related acute lung injury, the leading cause of death after transfusion therapy, activated platelets were described to induce the formation of NETs[ 80 ].
In another study, platelet were suggested to bind to neutrophils in the lungs, with subsequent activation of neutrophils by platelet toll-like receptor TLR 4[ 87 ]. In order to induce this response, cell-cell contact between the apoptotic neutrophil and monocytes was required[ 78 ].
After leaving the bone marrow, the neutrophil becomes part of one of the two compartments found in blood: the circulating pool and the marginated pool. The circulating pool consists of neutrophils flowing freely through vascular spaces and the marginated pool consists of neutrophils adhered to the endothelium of capillaries and post capillary venules, often in the lung, liver and spleen[ 15 ].
Already in , Cohnheim observed cells in a marginal position along venule walls. This gave rise to the hypothesis that a marginated pool should exist. Next, it was found that leukocytes circulate freely in the blood, then adhere to the vascular endothelium, especially in sites where the blood flow is slow and then re-enter the circulation in a continuously exchanging process[ 88 ].
The relative size of the marginated and circulating pool however, can be affected during exercise or induced by adrenaline or drugs Figure 3. It has been suggested that during infection the marginated pool is minimized, while the freely circulating pool becomes larger[ 89 ].
The marginated pool consists of neutrophils adhered to the endothelium of capillaries and postcapillary venules, often in the lung, liver and spleen. The bone marrow has also been suggested as a margination site[ 90 ]. Margination means a prolonged transit through these specific organs, resulting in an intravascular neutrophil pool. The lung has been a controversial margination site.
Some data suggest that the lung is the predominant site of margination[ 91 ], but this has been called into question by others[ 92 ]. Interestingly, different neutrophil types localized in different organs[ 93 ]. Suratt et al [ 93 ] showed that mature peripheral blood neutrophils localize to the liver, bone marrow and to a lesser extent to the spleen.
Younger marrow-derived neutrophils prefer to home back to the bone marrow, a process that will be described below, and inflammatory peritoneal neutrophils prefer the liver and the lungs. The biodistribution of inflammatory neutrophils might be non-comparable with homeostatic conditions as these neutrophils are different in surface expression of receptors and in functioning. Apoptotic neutrophils are not detected in normal circulation, so the need for an efficient removal system is evident, as 10 11 neutrophils are believed to be produced and removed every day.
Surface receptor expression is highly dynamic upon infection, but receptor expression also changes upon aging. As neutrophils become senescent, expression of a receptor for chemotaxis, CXCR2, decreases, while the expression of a chemokine receptor, CXCR4, increases[ 77 , 94 ].
CXCR4 thus is not only a signal to retain neutrophils in the bone marrow, but is also acting on homing senescent cells to the marrow for destruction. CXCR4 expression is up regulated just before apoptosis and after homing to the bone marrow, the neutrophils will undergo apoptosis and are subsequently phagocytosed by stromal macrophages, which are present in the hematopoietic cords[ 73 , 95 ].
Furze et al [ 96 ] showed that in mice, about one third of In-labeled neutrophils were cleared via bone marrow stromal macrophages. Before, stromal macrophages were only known for the removal of cellular debris and non-productive B cells[ 97 ].
Homing neutrophils must actively migrate through the bone marrow endothelium, a process that is not possible for apoptotic neutrophils. Neutrophils also home back to the bone marrow while the liver and spleen also remove circulating neutrophils.
Furze et al [ 96 ] showed that phagocytosis of neutrophils in the bone marrow stimulates G-CSF production which in turn induces neutrophil production in the bone marrow.
Interestingly, when apoptotic neutrophils are phagocytosed by reticular endothelial macrophages in the spleen and liver or by macrophages on a site of infection, the production of G-CSF is suppressed to limit the inflammation[ 98 ].
This way, via the up regulation of G-CSF production directly in the bone marrow, the production of new neutrophils can be tightly regulated. So if neutrophils are already apoptotic in circulation, the spleen and liver will clear them.
On the other hand, senescent neutrophils can migrate back into the bone marrow and will be cleared there, as a positive feedback loop for neutrophil production. To determine whether homing neutrophils can return to circulation, isolated neutrophils from the bone marrow and peripheral blood of mice were labeled and injected back into the mice[ 92 ]. About 20 percent of labeled mature bone marrow neutrophils remobilized during an inflammatory response.
However, homed bone marrow peripheral neutrophils could not be remobilized in response to inflammation. Therefore, the bone marrow could be seen as a site for clearance. In addition, this study also showed that infused marrow neutrophils may be remobilized. It would be very interesting to further investigate the recirculating potential of mature neutrophils, as this can greatly influence our understanding of neutrophil kinetics.
The kinetics of neutrophil production, the amount of cells that are produced each day, is measured as a rate of turnover of neutrophils in the blood. Blood neutrophil turnover has been determined by labeling neutrophils with [ 32 P] DFP di-isopropyl fluorophosphate and has been described to be about 1.
Marrow neutrophil production has been determined from the number of neutrophils in the post mitotic pool, divided by their transit time the appearance in circulating neutrophils of injected 3 H-thymidine Figure 4. The post mitotic pool consists of about 5. The marrow neutrophil production has therefore been calculated to be 0.
This amount corresponds to the calculated neutrophil turnover in blood. However, when cells were labeled with di-isopropylfluorophosphate- 32 P, a larger turnover of neutrophils was found.
Care should thus be taken with calculations and amounts, as they depend on the method to label cells[ 14 ]. The different maturation stages all have different kinetics, which are studied in vivo and in vitro using radioisotopic labeling. These studies indicate that between the myeloblast and the myelocyte stages, approximately five cell divisions occur[ , ]. Myelocytes probably undergo about three cell divisions, indicating the major expansion of the neutrophil pool to be at the myelocyte stage.
These radionuclide studies suggest that the transit time from myeloblast to myelocyte takes about h, divided over the different myelocyte stages Figure 2.
The transition from myelocyte to blood neutrophil takes about h, indicating a total time of approximately 12 d from precursor to mature neutrophil[ ]. During infection, transition time from myelocyte to blood neutrophil can be shortened to 48 h.
Following production, mature post mitotic neutrophils approximately 10 11 cells will remain in the bone marrow for d[ 14 , ]. In response to infection, the storage pool in the bone marrow will be used as source of neutrophils for blood neutrophilia[ ]. In conclusion, before a neutrophil leaves the bone marrow, it takes 17 d to be produced and maturated[ ].
The kinetics of neutrophils leaving the vascular compartment and their take-over by new neutrophils can easily be measured by labeling neutrophils and measure the transit time through the vascular compartment. When healthy individuals are injected with neutrophils, they leave the vascular compartment with a 7 h half life time[ 17 , ]. Using radiolabelled neutrophils and other analytical techniques, the neutrophil intravascular transit time has been measured for the liver, spleen and bone marrow, being respectively 2 and 10 min.
The intravascular transit time can be seen as the mean time taken for neutrophils to pass through the capillary bed of a specific organ. The influence of the marginated pool, homing back to the bone marrow and the kinetics in the spleen and liver on this transit time is unknown. As the regulation of neutrophil production and clearance is an important homeostatic mechanism and also involved in the development of systemic inflammatory states, it is of great importance that the kinetics of circulation and clearance are clear.
Now we know that not only the liver and spleen, but also the bone marrow clears neutrophils, and that the different organs clear different types of neutrophils[ 92 ]. But the function of neutrophils leaving the vascular compartment is largely unknown.
As described before, inflammatory neutrophils were found to have many more functions then only clearance of microbes. Possibly, neutrophils in marginated sites outside the vascular compartment, also have additional functions. There is growing evidence that to a certain extend neutrophils influence the adaptive immune response, either through pathogen shuttling to the lymph nodes[ ], through antigen presentation[ ], and through modulation of T helper responses[ ].
However, these described functions have still not been shown in vivo and also, are they neutrophil specific or do they occur as side effects of the functioning as a microbe-killer. Without signs of infection, neutrophils do not get activated and have no need to go into the tissues. They also do not exocytose their granules, meaning that they are not as harmful for the host as activated neutrophils.
The fate of these unactivated neutrophils is hard to investigate. Labeling neutrophils has revealed some of their fate, but labeling can also cause changes in the neutrophil e. However, the studies which labeled neutrophils and followed their route through the human body are still very useful in this context. In studies measuring neutrophil kinetics, different types of radioactive labeling have been used.
Furthermore, the label is only slightly or not at all attached to lymphocytes or monocytes[ ]. The effects of these radioactive labels on neutrophils have been studied by several authors, for example the effects on chemotactic responsiveness[ ]. Some labels are no longer in use, for example Na 2 51 CrO 4 and SnCl 2 -reduced 99m TcO 4 - , which showed less optimal results in the chemotactic responsiveness studies. Other labels are still used, for example 32 DFP or 3 H-thymidine.
For a long time, only in vitro labeling was possible, where neutrophils were isolated from a blood sample, which could easily stimulate the neutrophils. Upon stimulation, neutrophils release their granules and are altered in surface receptor expression, and although the labeling experiments have been improved hardly any research was done to assess the activation of neutrophils or the change in surface receptor expression due to labeling[ ].
Some authors claim that there is no difference in neutrophil activation, without showing the data. But as neutrophils are quick responders to differences in their homeostatic environment, in vitro , in vivo or in situ labeling can have tremendous effects on the cell, affecting the outcome of a study as well.
In mice, neutrophils were shown to have a half-life of 8 to 10 h when labeled in vivo [ ]. This shows that the methods for labeling can have an devastating effect on the outcome of the study. But unfortunately, extrapolation of mice experiments is often very difficult. In mice, neutrophils are not the main circulating white blood cell-type, they do not express the same receptors as human neutrophils for example there is a lack CXCR1 and also chemoattractant CXCL-8 does not exist in mice.
Therefore, care should be taken when mice are used for calculating neutrophil life spans. Most experiments done with in vitro labeling have not been repeated with in vivo labeling, meaning that some knowledge needs to be adjusted. Recently, Pillay et al [ 16 ] used 2 H 2 O, a new labeling method for labeling neutrophil pools in vivo , to calculate the rate of division of the mitotic pool in the bone marrow, the transit time of new neutrophils through the post mitotic pool and the delay in mobilization of neutrophils from the post mitotic pool to the blood.
They recalculated the life-span of neutrophils and found an average circulatory neutrophil lifespan of 5. However, there are also doubts concerning this report, as the previously used radioactive labels e. The new model is thought to lack the right temporal resolution to make these conclusions, as the mean value of the total life span of a neutrophil is in line with the previously described total life span[ ]. Also, the authors did not show that the deuterium was not reutilized in newly dividing neutrophil precursor, thereby possibly influencing the results[ 18 ].
Either one of these two numbers should be reconsidered. Interestingly, different maturation states of neutrophils are labeled by different radioactive labels. Warner and Athens compared the three most common radioactive labels in vitro until , 3 H-thymidine, 32 P-labeled sodium phosphate and 32 DFP, in their kinetics regarding the blood granulocyte radioactivity curves measured after administration[ ].
The component s in the granulocytes to which DFP binds is unknown as DFP binds many different esterases and proteolytic enzymes[ ]. When the blood kinetics of all three populations is compared, they are all three totally different: 32 DFP levels start high, where after the labeled neutrophils disappear in marginated pools and the level of 32 DFP declines. In our opinion in vivo labeling is the better method.
Isolating blood cells, processing and inject them again in the recipient can have dramatic effects on their life span. When leukemia patients are transfused with donated red blood cells after bone marrow transplantation, the half life on the donated red blood cells is dramatically reduced, leading to massive clearance of red blood cells.
The released iron due to this enhanced turnover is a well known complication of red cell transfusion[ ]. This indicates that even careful isolation of blood cells without any labeling has an impressive effect on their life span. A proper understanding of the lifespan and distribution of the neutrophil is very important, as the neutrophil can vary in phenotype and function with a longer lifespan, and the lifespan determines the need for influencing the neutrophil function in inflammatory diseases.
Further investigation of these different labeling techniques, their influence on neutrophil life span and the actual life span of a neutrophil are needed.
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