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Published online before print December 5, 2005
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* The Centre for Cardiovascular Sciences, Centre for Immune Regulation and the MRC Centre, The University of Birmingham, United Kingdom; and
Department of Vascular Surgery, University of Birmingham Hospital (Selly Oak), United Kingdom
1Correspondence: The Department of Physiology, The Medical School, The University of Birmingham, UK, B15 2TT. E-mail:g.e.rainger{at}bham.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.25%). RT neutrophils were distinct from naïve circulatory neutrophils (CD54low, CXCR1high) and naïve cells after activation with formyl-Met-Leu-Phe (CD54low, CXCR1low). It is important that the RT phenotype (CD54high, CXCR1low) is also distinct from tissue-resident neutrophils (CD54low, CXCR1low). Our results demonstrate that neutrophils can migrate in a retrograde direction across endothelial cells and suggest that a population of tissue-experienced neutrophils with a distinct phenotype and function are present in the peripheral circulation in humans in vivo.
Key Words: migration • recirculation • chronic inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neutrophils, conversely, are effector cells of the innate immune response and upon recruitment to tissue, are not generally thought to exhibit functional plasticity as a result of differentiation. Current understanding is that neutrophils are terminally differentiated, short-lived cells, which do not recirculate. Upon recruitment into tissue, they prosecute their immune functions aggressively and then die by apoptosis within the tissue stroma, their remains cleared by tissue macrophages [6 ]. To date, neutrophils have not been reported to emigrate out of tissues via the lymphatics. However, there have been suggestions from studies of experimental glomerular capillary injury in the rat that neutrophils can emigrate out of inflamed tissue, apparently returning to the circulation [7 ].
We have previously documented the fate of neutrophils during their recruitment by cytokine-stimulated EC in in vitro models of inflammation. We have modeled the recruitment of neutrophils into inflamed tissue by culturing EC in flattened glass capillaries (microslides), stimulating them for 4 h with the inflammatory cytokine tumor necrosis factor 
(TNF-) [8
] and examining how flowing neutrophils transmigrate across the endothelium. Using this dynamic, in vitro model, we have established that flowing neutrophils are captured by the receptors E- and P-selectin [9
] and that subsequent activation and transendothelial migration are promoted by CXC chemokine(s), which operate through the receptor CXCR2 [10
]. These observations have extended our knowledge of neutrophil behavior during the recruitment process but have been limited to studying interactions within the first 30 min following contact with EC. To examine the long-term fate of recruited neutrophils in our system, we established an assay that determined how neutrophils interact with EC for up to 24 h. We have already reported that cross-talk between recruited neutrophils and EC regulates the transcriptional activity of EC molecules such as vascular cell adhesion molecule-1 (CD106), required for the subsequent recruitment of mononuclear leukocytes [11
]. Here, we report that when transmigrated neutrophils are maintained in coculture with EC, they can reverse-transmigrate (RT) back across an EC monolayer in a manner similar to that described previously for monocytes [12
]. These RT neutrophils constitute a phenotypically and functionally differentiated population, with a characteristic phenotype (CD54high, CXCR1 low). It is important that similar cells are found in vivo in the blood of healthy donors and at significantly higher levels, in patients with chronic active inflammatory disease, suggesting that RT neutrophils are pathologically important and may play a role in the persistence of inflammation in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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EC isolation and coculture with neutrophils
Human umbilical vein EC (HUVEC) were isolated and cultured as described previously by Cooke et al. [8
]. HUVEC were cultured in microslides (flattened glass capillaries) or onto culture plastic in 25 cm2 dishes for 24 h until confluent, as described previously [8
]. EC monolayers in microslides or in culture flasks were activated with 100 U/ml recombinant human TNF (R&D Systems, Abingdon, UK) for 4 h. TNF was washed from the system, and purified neutrophils were introduced at concentrations sufficient to yield six neutrophils/EC. After 1 h, nonadherent cells were washed from the EC surface. Neutrophils and EC were then cocultured for 24 h in the continued presence of TNF. Neutrophils were washed away to waste by automated medium changes in the microslide system but could be harvested from the EC surface in culture flasks at the conclusion of coculture.
In some experiments, EC were subcultured onto transwell filters, which had 3 µm pores (Becton Dickinson, Franklin Lakes, NJ). EC monolayers were treated as per microslide experiments with the exception that after 1 h of neutrophil incubation, nonadherent neutrophils and cells adherent to the apical surface of the monolayer were removed with three washes with culture medium. Neutrophils and EC were then cocultured for 20 h, and cells that had migrated through the EC and across the porous membrane were collected from the lower chamber of the transwell system. RT neutrophils were harvested from the apical surface of the EC monolayer using a single wash in culture medium.
In some experiments, antibodies against ß1 (MAB13; Becton Dickinson, Cowley, UK), ß2 (6.5E; gift of Dr. Martyn Robinson, CellTech, Slough, UK), or ß3 integrins (SZ21; Immunotech, Marseille, France) were included in the coculture medium at 10 ug/ml. These reagents have been demonstrated to block integrin function in previous studies.
Assessment of neutrophil apoptosis
For assessment by nuclear morphology, neutrophils were centrifuged onto glass slides for 3 min at 10 g in a Shandon Cytospin II (Scientific Instruments, South Trentham, UK) and stained with Diff Quik. Cells with one or more darkly stained, condensed nuclear fragment(s) were deemed apoptotic (see Fig. 2
). Apoptosis by caspase-3 activity was assessed using a commercially available kit (R&D Systems), according to the manufacturer’s instructions, and quantified by Fluoroskan Ascent microplate reader (Labsystems: Affinity Sensors, Cambridge, UK). Assessment of apoptosis by mitochondrial membrane permeability was assessed using 40 nM DiOC6 with fluorescence intensity analyzed using a Coulter XL flow cytometer (Beckman Coulter, High Wycombe, UK) and WinMDI software.
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for 4 h and were incorporated into a flow-based adhesion assay [9
, 10
]. Freshly isolated peripheral blood neutrophils or RT neutrophils (1x106/ml) were perfused for 3 min at a wall shear stress of 0.1 Pa. Nonadherent cells were washed out, and after15 min (3 min of cell perfusion and 12 min of wash), five randomly selected fields were recorded via videomicroscopy. Adherent cells were counted in the video records and were categorized as rolling (phase bright and tumbling slowly across the EC); activated (phase bright, shape changed and stably attached to the apex of the EC); and transmigrated (phase dark and migrated through the EC monolayer).
Measurement of neutrophil regidity
The ability of neutrophil suspensions (106 cells/ml) to pass through rigid filters was measured using a St. George’s filtrometer (Carri-Med Ltd., Dorking, UK) and nucleopore polycarbonate filters (pore diameter=5 µm; Hemafil, Nucleopore Corp., Pleasanton, CA) as described previously [13
]. In this device, a constant hydrostatic pressure of 10 cm H2O drew the cell suspension through a filter. The flow rate was measured over a 60-µl test vol after 750 µl sample had been filtered, relative to the flow rate of cell-free buffer, which was measured first. In general, the relative flow rate (rFR) depends on the average resistance to flow of the main population of neutrophils and on the size of any subpopulation able to block pores [14
]. Typically, if freshly isolated neutrophils were treated with 10–7M bacterial formyl-Met-Leu-Phe (fMLP) peptide (Sigma), rFR decreased to <0.01, which indicated that the filter had become blocked effectively.
Measurement of superoxide anion production
Superoxide production, by freshly isolated or RT neutrophils, was measured using a colorimetric assay reliant on the reduction of cytochrome C as described [15
]. Prior to assay, neutrophils were untreated or activated with fMLP (10–7 M, Sigma). Superoxide production was measured at 10-min intervals for 1 h and expressed as nM/106 cells.
Identification of RT neutrophils by flow cytometry
Packed cells (50 µl) from whole blood or RT neutrophils were incubated on ice with appropriate antibodies. Expression of chemokine receptors was measured using phycoerthyrin (PE) anti-CXCR1 or CXCR2 together with biotinylated anti-CD54 (all R&D Systems). Nonspecific staining was tested using appropriate isotype controls (Dako, Copenhagen, Denmark). Cells were then washed in ice-cold buffer and incubated with Streptavidin-PE/Cy5 (Dako) to detect the biotinylated antibody. After washing, cells were fixed with fluorescein-activated cell sorter lysing solution (BD Biosciences, UK), and the number of RT neutrophils was quantified using a BD EPICS XL cytometer (Beckman Coulter). RT neutrophils were analyzed by identifying the neutrophil population by forward and side light scatter and then gating on the neutrophil population, which fell outside the distribution parameters for CD54 and CXCR1 in the control population. An anti-human CD3-fluorescein isothiocyanate (Dako) antibody was substituted to quantify natural killer (NK) cells. Details of antibody clone designations can be found in Table 1
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated EC in vitro-stimulated EC grown in glass capillaries, 
60% of the adherent cells transmigrated into the subendothelial compartment within the first hour (Fig. 1A
and 1B
) as described previously [8
9
10
]. However, when we attached the capillaries to a flow system that perfused the coculture with buffer for 1 min every hour, we found that the transmigrated neutrophils disappeared from the system in a biphasic manner until only 5% of the original migrated neutrophils was left after 20 h (Fig. 1B
and 1C)
. These results suggested to us that the neutrophils had re-emerged from under the endothelial monolayer, lost their adhesive contact, and been washed away.
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RT neutrophils are functionally distinct from naive circulating neutrophils
During inflammation, naïve resting neutrophils are recruited into tissues by EC that have been activated by inflammatory cytokines at sites of inflammation [1
, 2
]. To investigate whether RT neutrophils were functionally competent in this respect, we determined whether they could be recruited from flow by a second monolayer of EC that had been stimulated with TNF-. Neither freshly isolated nor RT neutrophils bound to unstimulated EC (Fig. 3A
), but both adhered to stimulated EC. Flowing RT neutrophils adhered to TNF-stimulated EC with a reduced efficiency compared with freshly isolated cells (Fig. 3A)
. When we examined further the behavior of the freshly isolated and RT neutrophils on activated endothelium, we observed that although 60% of freshly isolated cells transmigrated across the EC monolayer, less than 1% RT neutrophils could transmigrate (Fig. 3B)
. Virtually all of the RT neutrophils were stationary and adherent on the EC surface and did not roll (Fig. 3B)
. These results imply that RT neutrophils are unlikely to be able to re-enter an inflamed tissue.
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A major function of activated neutrophils is the production of cytotoxic agents such as reactive oxygen species (ROS). We measured the ability of RT neutrophils to generate a superoxide burst compared with freshly isolated and tissue plastic-cultured cells. Neutrophils cultured on plastic contained a significant population of apoptotic cells and were unable to generate a superoxide burst, even when stimulated with fMLP. Freshly isolated neutrophils and RT neutrophils generated significant levels of superoxide when they were activated with fMLP (Fig. 3D) . In addition, RT neutrophils generated greater amounts of superoxide than freshly isolated cells, indicating that they were functionally primed for an enhanced response. Overall, these results suggest that although RT neutrophils would be unlikely to enter tissue at secondary sites of inflammation, they might become adherent or physically trapped in microvessels and would then be capable of generating a powerful oxidative burst.
Reverse-migrated neutrophils have a distinct surface phenotype
We determined if RT neutrophils had a surface phenotype, which would allow them to be distinguished from other neutrophils by immunofluorescent staining and flourimetry. Initially, we compared the expression of a wide range of surface receptors, including adhesion molecules and chemokine receptors, on RT neutrophils harvested form EC monolayers cultured on plastic or on freshly isolated neutrophils from healthy donors (Table 1) . The expression of most receptors was unaltered by RT or as in the case of V and 4 integrin, were so variable in expression that large changes failed to show significance. However for some markers, there were clear differences. The expression of the ß2 integrin (CD18) was increased, and L-selectin (CD62L) was decreased on RT cells in a manner consistent with neutrophil activation [12
] (Table 1)
. It is interesting that intercellular adhesion molecule-1 (ICAM-1; CD54) was increased dramatically, whereas expression of the chemokine receptors CXCR1 and CXCR2 was decreased on RT compared with freshly isolated neutrophils (Table 1
and Fig. 4
). These changes in expression were not a result of a nonspecific, general activation of the neutrophils, as ICAM-1 up-regulation was confined to RT neutrophils and did not occur with fMLP stimulation of freshly isolated cells (Fig. 4)
. Therefore, alterations in CD54 and CXCR1 allowed us to define a unique phenotype for RT neutrophils (CD54 high, CXCR1 low), which was distinct from freshly isolated (CD54low, CXCR1high) or fMLP-activated (CD54low, CXCR1 low) neutrophils.
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RT neutrophils also had a phenotype that was distinct from once migrated cells in vivo. Neutrophils from the synovial fluid of RA patients are recruited from the vasculature, traverse the synovial panus, and enter the synovial fluid, i.e., cells that have crossed an EC barrier only once. Synovial fluid neutrophils had the same phenotype as once migrated neutrophils from the lower well of our transwell experiments (CXCR1low, CD54low; Fig. 4 ). These were distinct from circulating neutrophils in the blood of RA patients, which did not demonstrate elevated levels of CD54 (Fig. 4) .
Neutrophils with the phenotype of reverse-migrated cells are found in the peripheral blood
Having identified a unique phenotype for RT neutrophils, we wondered if cells with this phenotype could be observed in peripheral blood in vivo. To develop an assay that could accurately quantify these cells in vivo, freshly drawn blood was "spiked" with RT neutrophils generated in vitro at a final concentration of 0%, 1%, or 10% of the donor population. Using the phenotype CD54high, CXCR1low, we could detect this population easily in the mixture. The assay reported accurately the number of spiked RT neutrophils added (Fig. 5A
5B
5C
).
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0.25% of all circulating neutrophils (Fig. 5)
. To ensure that these cells shared more of the phenotypic character of our in vitro RT cells, we also verified that CD54high, CXCR1low cells showed a significant reduction in CXCR2 (data not shown). To verify that this population represented only neutrophils, we back-gated on the population defined as neutrophils by forward and side light scatter and found that CD54high, CXCR1low cells were confined within this neutrophil gate. In addition, we confirmed that CD54high, CXCR1low cells were negative for CD3 and CD56, thereby excluding the possibility that these cells were contaminating NK cells (data not shown). Finally, we confirmed that cells in this gate were positive for CD16b, a well-recognized discriminator of neutrophils (mean fluorescence intensity for staining with CD16b or irrelevant control was 32±7 and 3±0.1, respectively; P=0.003 by paired t-test).
In patients with active RA (n=5), we could also identify a distinct population of cells with a phenotype consistent with those that had reverse-migrated in vitro (Fig. 5D)
. On average, recirculating RT neutrophils constituted 1% of the pool of blood neutrophils in these patients (Fig. 5F)
. The increased population of RT neutrophils was not confined to RA. CD54high, CXCR1low cells were also detectable in the blood of patients undergoing elective repair of aortic aneurysm, and severe atherosclerotic disease of the aorta (Fig. 5E
and 5F
; n=3), constituting 1% of the circulating population of blood neutrophils. Thus, cells with the phenotype of RT neutrophils, are not only found in the peripheral blood of healthy individuals but at increased numbers in the peripheral blood of patients with chronic inflammatory arthritis and atherosclerosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Attempts to define distinct subsets of circulating neutrophils in the peripheral blood of humans have proven to be difficult, although alterations in the function of circulating neutrophils have been reported in response to priming during inflammation or infection (e.g., refs. [7 , 16 17 18 ]). Recently, three different neutrophil subsets with distinct functions, reflected by activation-dependent changes in cytokine/chemokine production, macrophage activation, and expression of Toll-like receptors, have been described in mice, which exhibited different susceptibilities to infection with methicillin-resistant Staphylococcus aureus [19 ]. However, this profile of neutrophil subset distribution is established during haematopoiesis and does not reflect the capacity of neutrophils to respond to activation in an adaptive manner. Conversely, we show that neutrophils can adopt a unique phenotype that is associated with functional maturation as well as being marked by surface alterations imposed during the process of RT. Thus, we believe that this is the first description of a distinct population of circulating neutrophils, which have differentiated in a manner independent of the process of hematopoietic maturation.
We postulate that in addition to in situ phagocytic clearance of neutrophils, there could be an additional mechanism for clearing neutrophils recruited to "normal" tissue during immune surveillance. For example, in all vertebrates, neutrophils and lymphocytes are recruited continuously to mucosal tissues, such as the gut, lung, and peritoneum in the absence of inflammation or infection [20 21 22 23 ]. Neutrophils can constitute up to 10% of this leukocytic infiltrate in healthy individuals, although this can rise dramatically to over 50% upon initiation of inflammation [21 22 23 ]. We suggest that during immune surveillance, these cells are cleared, at least in part, by a counter-traffic from the tissues back into the circulation. They would then represent a small fraction of the continuously renewed, circulatory population.
Once RT neutrophils have returned to the circulation, they are likely to be removed efficiently by the specialized organs of the reticuloendothelial cell system (e.g., the spleen and the liver), which have a population of macrophages in direct contact with flowing blood. The important role of the liver in regulating the numbers of circulating neutrophils has been confirmed by recent studies, which demonstrated neutrophil sequestration through P-selectin [24 ]. The loss of the chemokine receptors CXCR1 and CXCR2 and the inability of RT neutrophils to transmigrate across inflamed EC indicate that they are unlikely to be able to enter tissues at sites of inflammation. In addition, RT neutrophils are relatively rigid, a physical characteristic that might retard their passage through the microvasculature of the tissues and prolong contact with the sinusoids of the reticuloendothelial cell system. It is interesting that ICAM-1 (CD54) is a high-affinity ligand for the ß2 integrins of macrophages resident in the reticuloendothelial cell system, and the large increase in ICAM-1 expression on RT neutrophils could promote efficient removal of these cells from the circulation.
We have also reported a marked (100%) increase in the number of RT neutrophils in disease states with a chronic inflammatory component. We postulate that the increased size of a population of functionally primed, recirculating neutrophils, with a predilection for mechanical or adhesive entrapment in the vasculature, could contribute to pathology, for example, in rheumatoid vasculitis [25 ]. The etiology of this inflammatory disease of the blood vessel wall is poorly understood; however, it is mediated by inappropriately activated neutrophils, and the presence of an expanded population of RT neutrophils is suggestive of a link between these cells and disseminated vasculopathies in rheumatoid disease.
A population of recirculating neutrophils may also be sensitive to systemic, inflammatory mediators, released, for example, during surgery [26 , 27 ] for cardivascular disease. These cells could again become mechanically entrapped in the microvasculature of major organs (most likely in the pulmonary circulation). By initiating localized ischaemia in association with the elaboration of ROS, activated RT neutrophils could contribute to distant organ (pulmonary) damage and perpetuate multiorgan failure, an unwanted outcome of major surgery, which is more prevalent in patients with established chronic inflammatory disease.
In conclusion, the existence of a recirculting population of tissue-experienced neutrophils has been postulated [7 ] but has not been demonstrated formally. In the current study, we show that neutrophils can migrate in a retrograde direction across an EC monolayer in vitro, that these cells are altered functionally and in terms of their surface phenotype, and that a phenotypically identical population circulates in the blood. The pathophysiological implications of RT of neutrophils remain to be elucidated, but their increased incidence in chronic inflammatory diseases suggests that they are likely to be associated with the pathology of these diseases.
Received September 2, 2005; revised October 14, 2005; accepted October 18, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Adhes. Commun. 6,491-501[Medline]
receptors CD16 and CD64 on polymorphonuclear neutrophils: data from patients with severe bacterial infections and lipopolysaccharide-exposed cells Shock 19,5-12[CrossRef][Medline]This article has been cited by other articles:
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  M. W. Johansson, E. A. B. Kelly, W. W. Busse, N. N. Jarjour, and D. F. Mosher Up-Regulation and Activation of Eosinophil Integrins in Blood and Airway after Segmental Lung Antigen Challenge J. Immunol., June 1, 2008; 180(11): 7622 - 7635. [Abstract] [Full Text] [PDF]
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 P. F. Bradfield, S. Nourshargh, M. Aurrand-Lions, and B. A. Imhof JAM Family and Related Proteins in Leukocyte Migration (Vestweber Series) Arterioscler. Thromb. Vasc. Biol., October 1, 2007; 27(10): 2104 - 2112. [Abstract] [Full Text] [PDF]
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N. Farahi, A. S. Cowburn, P. D. Upton, J. Deighton, A. Sobolewski, E. Gherardi, N. W. Morrell, and E. R. Chilvers Eotaxin-1/CC Chemokine Ligand 11: A Novel Eosinophil Survival Factor Secreted by Human Pulmonary Artery Endothelial Cells J. Immunol., July 15, 2007; 179(2): 1264 - 1273. [Abstract] [Full Text] [PDF]
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 J. R. Mathias, B. J. Perrin, T.-X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish J. Leukoc. Biol., December 1, 2006; 80(6): 1281 - 1288. [Abstract] [Full Text] [PDF]
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