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1.  Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF 
The Journal of Experimental Medicine  2014;211(7):1307-1314.
Adherent neutrophils responding to chemoattractants release TNF in proximity of endothelial cell junctions to mediate microvascular leakage.
Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB4, C5a, and KC. Further, in TNFR−/− mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in life-threatening acute edema.
PMCID: PMC4076577  PMID: 24913232
2.  Shed syndecan-2 inhibits angiogenesis 
Journal of Cell Science  2014;127(21):4788-4799.
Angiogenesis is essential for the development of a normal vasculature, tissue repair and reproduction, and also has roles in the progression of diseases such as cancer and rheumatoid arthritis. The heparan sulphate proteoglycan syndecan-2 is expressed on mesenchymal cells in the vasculature and, like the other members of its family, can be shed from the cell surface resulting in the release of its extracellular core protein. The purpose of this study was to establish whether shed syndecan-2 affects angiogenesis. We demonstrate that shed syndecan-2 regulates angiogenesis by inhibiting endothelial cell migration in human and rodent models and, as a result, reduces tumour growth. Furthermore, our findings show that these effects are mediated by the protein tyrosine phosphatase receptor CD148 (also known as PTPRJ) and this interaction corresponds with a decrease in active β1 integrin. Collectively, these data demonstrate an unexplored pathway for the regulation of new blood vessel formation and identify syndecan-2 as a therapeutic target in pathologies characterised by angiogenesis.
PMCID: PMC4215719  PMID: 25179601
Angiogenesis; Inflammation; Syndecan; Integrin; Endothelial cell migration
3.  ICAM-2 facilitates luminal interactions between neutrophils and endothelial cells in vivo 
Journal of Cell Science  2014;127(3):620-629.
Intercellular adhesion molecule 2 (ICAM-2) is expressed on endothelial cells (ECs) and supports neutrophil extravasation. However, the full details of its role remain unknown, and the present study investigates the functional mechanisms of ICAM-2 in neutrophil–endothelial-cell interactions. Our initial studies showed expression of ICAM-2 at both EC junctions and on the EC body. In line with the observed expression profile analysis of neutrophil–vessel-wall interactions using real-time in vivo confocal microscopy identified numerous functional roles for ICAM-2 within the vascular lumen and at the stage of neutrophil extravasation. Functional or genetic blockade of ICAM-2 significantly reduced neutrophil crawling velocity, increased frequency of crawling with a disrupted stop-start profile, and prolonged interaction of neutrophils with EC junctions prior to transendothelial cell migration (TEM), collectively resulting in significantly reduced extravasation. Pharmacological blockade of the leukocyte integrin MAC-1 indicated that some ICAM-2-dependent functions might be mediated through ligation of this integrin. These findings highlight novel roles for ICAM-2 in mediating luminal neutrophil crawling and the effect on subsequent levels of extravasation.
PMCID: PMC4007766  PMID: 24317296
Intercellular adhesion molecule 2; ICAM-2; Leukocyte; Neutrophil; Extravasation; Intravascular crawling
4.  Endothelial cell junctional adhesion molecule C plays a key role in the development of tumors in a murine model of ovarian cancer 
The FASEB Journal  2013;27(10):4244-4253.
Junctional adhesion molecule C (JAM-C) is a transmembrane protein with significant roles in regulation of endothelial cell (EC) functions, including immune cell recruitment and angiogenesis. As these responses are important in promoting tumor growth, the role of EC JAM-C in tumor development was investigated using the ID8 syngeneic model of ovarian cancer. Within 10–15 wk, intraperitoneally injected ID8 cells form multiple tumor deposits and ascites that resemble human high-grade serous ovarian cancer. Compared to wild-type mice, survival in this model was increased in EC JAM-C knockouts (KOs; 88 vs. 96 d, P=0.04) and reduced in EC JAM-C transgenics (88 vs. 78.5 d, P=0.03), mice deficient in or overexpressing EC JAM-C, respectively. While tumor growth was significantly reduced in EC JAM-C KOs (87% inhibition at 10 wk, P<0.0005), this was not associated with alterations in tumor vessel density or immune cell infiltration. However, tumor microvessels from EC JAM-C-deficient mice exhibited reduced pericyte coverage and increased vascular leakage, suggesting a role for EC JAM-C in the development of functional tumor vessels. These findings provide evidence for a role for EC JAM-C in tumor growth and aggressiveness as well as recruitment of pericytes to newly formed blood vessels in a model of ovarian cancer.—Leinster, D. A., Colom, B., Whiteford, J. R., Ennis, D. P., Lockley, M., McNeish, I. A., Aurrand-Lions, M., Chavakis, T., Imhof, B. A., Balkwill, F. R., Nourshargh, S. Endothelial cell junctional adhesion molecule C plays a key role in the development of tumors in a murine model of ovarian cancer.
PMCID: PMC3819510  PMID: 23825230
pericytes; angiogenesis; vascular development; immune cell infiltrate
5.  Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves 
The FASEB Journal  2012;26(3):1064-1076.
Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.—Colom, B., Poitelon, Y., Huang, W., Woodfin, A., Averill, S., Del Carro, U., Zambroni, D., Brain, S. D., Perretti, M., Ahluwalia, A., Priestley, J. V., Chavakis, T., Imhof, B. A., Feltri, M. L., Nourshargh, S. Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves.
PMCID: PMC3370675  PMID: 22090315
adhesion molecules; tight junctions; peripheral nerves
6.  Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo 
The Journal of Experimental Medicine  2012;209(6):1219-1234.
After transendothelial cell migration, neutrophils actively crawl along pericyte processes before exiting the venular wall via selected gaps between adjacent pericytes.
Neutrophil transmigration through venular walls that are composed of endothelial cells (ECs), pericytes, and the venular basement membrane is a key component of innate immunity. Through direct analysis of leukocyte–pericyte interactions in inflamed tissues using confocal intravital microscopy, we show how pericytes facilitate transmigration in vivo. After EC migration, neutrophils crawl along pericyte processes to gaps between adjacent pericytes in an ICAM-1–, Mac-1–, and LFA-1–dependent manner. These gaps were enlarged in inflamed tissues through pericyte shape change and were used as exit points by neutrophils in breaching the venular wall. The findings identify previously unknown roles for pericytes in neutrophil transmigration in vivo and add additional steps to the leukocyte adhesion cascade that supports leukocyte trafficking into sites of inflammation.
PMCID: PMC3371725  PMID: 22615129
7.  Neutrophil heterogeneity in health and disease: a revitalized avenue in inflammation and immunity 
Open Biology  2012;2(11):120134.
Leucocytes form the principal cellular components of immunity and inflammation, existing as multiple subsets defined by distinct phenotypic and functional profiles. To date, this has most notably been documented for lymphocytes and monocytes. In contrast, as neutrophils are traditionally considered, to be short-lived, terminally differentiated cells that do not re-circulate, the potential existence of distinct neutrophil subsets with functional and phenotypic heterogeneity has not been widely considered or explored. A growing body of evidence is now challenging this scenario, and there is significant evidence for the existence of different neutrophil subsets under both physiological and pathological conditions. This review will summarize the key findings that have triggered a renewed interest in neutrophil phenotypic changes, both in terms of functional implications and consequences within disease models. Special emphasis will be placed on the potential pro- and anti-inflammatory roles of neutrophil subsets, as indicated by the recent works in models of ischaemia–reperfusion injury, trauma, cancer and sepsis.
PMCID: PMC3513838  PMID: 23226600
neutrophil; neutrophil subset; neutrophil phenotype; inflammation; neutrophil plasticity
8.  CD31 Exhibits Multiple Roles in Regulating T Lymphocyte Trafficking In Vivo 
The role of CD31, an Ig-like molecule expressed by leukocytes and endothelial cells (ECs), in the regulation of T lymphocyte trafficking remains contentious. Using CD31-deficient mice, we show that CD31 regulates both constitutive and inflammation-induced T cell migration in vivo. Specifically, T cell:EC interactions mediated by CD31 molecules are required for efficient localization of naive T lymphocytes to secondary lymphoid tissue and constitutive recirculation of primed T cells to nonlymphoid tissues. In inflammatory conditions, T cell:EC CD31-mediated interactions facilitate T cell recruitment to Ag-rich sites. However, endothelial CD31 also provides a gate-keeping mechanism to limit the rate of Ag-driven T cell extravasation. This event contributes to the formation of Ag-specific effector T cell infiltrates and is induced by recognition of Ag on the endothelium. In this context, CD31 engagement is required for restoring endothelial continuity, which is temporarily lost upon MHC molecule ligation by migrating cognate T cells. We propose that integrated adhesive and signaling functions of CD31 molecules exert a complex regulation of T cell trafficking, a process that is differentially adapted depending on cell-specific expression, the presence of inflammatory conditions and the molecular mechanism facilitating T cell extravasation.
PMCID: PMC3496211  PMID: 22966083
9.  The spatiotemporal localization of JAM-C following sciatic nerve crush in adult rats 
Brain and Behavior  2012;2(4):402-414.
JAM-C is a junctional adhesion molecule, enriched at tight junctions on endothelial and epithelial cells, and also localized to Schwann cells at junctions between adjoining myelin end loops. The role of JAM-C following peripheral nerve injury (PNI) is currently unknown. We examined the localization of JAM-C after sciatic nerve crush injury in adult rats. JAM-C immunoreactivity was present in paranodes and incisures in sham surgery control nerve, but distal to the crush injury significantly decreased at three and 14 days. JAM-C was re-expressed at 28 days and, by 56 days, was significantly increased in the distal nerve compared to controls. In a 7-mm length of sciatic nerve sampled distal to the crush site, the densities of JAM-C immunoreactive paranodes increased in the distal direction. Conversely, the densities of JAM-C immunoreactive incisures were highest immediately distal to the crush site and decreased in the more distal direction. Further analysis revealed a strong correlation between JAM-C localization and remyelination. Fifty-six days after crush injury, greater densities of JAM-C paranodes were seen compared to the nodal marker jacalin, suggesting that paranodal JAM-C precedes node formation. Our data are the first to demonstrate a potential role of JAM-C in remyelination after PNI.
PMCID: PMC3432963  PMID: 22950044
JAM-C; paranodes; peripheral nerve injury; remyelination; Schwann cells
10.  Primed T Cell Responses to Chemokines Are Regulated by the Immunoglobulin-Like Molecule CD31 
PLoS ONE  2012;7(6):e39433.
CD31, an immunoglobulin-like molecule expressed by leukocytes and endothelial cells, is thought to contribute to the physiological regulation T cell homeostasis due to the presence of two immunotyrosine-based inhibitory motifs in its cytoplasmic tail. Indeed, loss of CD31 expression leads to uncontrolled T cell-mediated inflammation in a variety of experimental models of disease and certain CD31 polymorphisms correlate with increased disease severity in human graft-versus-host disease and atherosclerosis. The molecular mechanisms underlying CD31-mediated regulation of T cell responses have not yet been clarified. We here show that CD31-mediated signals attenuate T cell chemokinesis both in vitro and in vivo. This effect selectively affects activated/memory T lymphocytes, in which CD31 is clustered on the cell membrane where it segregates to the leading edge. We provide evidence that this molecular segregation, which does not occur in naïve T lymphocytes, might lead to cis-CD31 engagement on the same membrane and subsequent interference with the chemokine-induced PI3K/Akt signalling pathway. We propose that CD31-mediated modulation of memory T cell chemokinesis is a key mechanism by which this molecule contributes to the homeostatic regulation of effector T cell immunity.
PMCID: PMC3378580  PMID: 22724015
11.  Expression and Function of Junctional Adhesion Molecule–C in Myelinated Peripheral Nerves 
Science (New York, N.Y.)  2007;318(5855):1472-1475.
JAM-C is an adhesion molecule that is expressed on cells within the vascular compartment and epithelial cells and, to date, has been largely studied in the context of inflammatory events. Using immunolabeling procedures in conjunction with confocal and electron microscopy, we show here that JAM-C is also expressed in peripheral nerves and that this expression is localized to Schwann cells at junctions between adjoining myelin end loops. Sciatic nerves from JAM-C–deficient [having the JAM-C gene knocked out (KO)] mice exhibited loss of integrity of the myelin sheath and defective nerve conduction as indicated by morphological and electrophysiological studies, respectively. In addition, behavioral tests showed motor abnormalities in the KO animals. JAM-C was also expressed in human sural nerves with an expression profile similar to that seen in mice. These results demonstrate that JAM-C is a component of the autotypic junctional attachments of Schwann cells and plays an important role in maintaining the integrity and function of myelinated peripheral nerves.
PMCID: PMC3299566  PMID: 18048693
12.  Junctional adhesion molecule-C (JAM-C) regulates polarized neutrophil transendothelial cell migration in vivo 
Nature immunology  2011;12(8):761-769.
Neutrophil migration into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarised migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial cell migration; TEM) in a luminal to abluminal direction. Using real-time confocal imaging we report that neutrophils can exhibit disrupted polarised TEM (“hesitant” and “reverse”) in vivo. These events were noted in inflammation following ischemia-reperfusion injury, characterised by reduced expression of junctional adhesion molecule C (JAM-C) from EC junctions, and were enhanced by EC JAM-C blockade or genetic deletion. The results identify JAM-C as a key regulator of polarised neutrophil TEM in vivo and suggest that reverse TEM neutrophils can contribute to dissemination of systemic inflammation.
PMCID: PMC3145149  PMID: 21706006
13.  Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148 
Molecular Biology of the Cell  2011;22(19):3609-3624.
The proteoglycan syndecan-2 is a novel ligand for the tyrosine phosphatase receptor CD148, an interaction that stimulates a signaling pathway leading to integrin-mediated cell adhesion. The pathway involves SRC and PI3 kinases and is an example of cell surface receptor cross-talk influencing integrin-mediated cellular processes.
Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is β1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine phosphatase receptor CD148 is shown to be a key intermediary in cell adhesion to S2ED, with downstream β1 integrin–mediated adhesion and cytoskeletal organization. We show that S2ED is a novel ligand for CD148 and identify the region proximal to the transmembrane domain of syndecan-2 as the site of interaction with CD148. A mechanism for the transduction of the signal from CD148 to β1 integrins is elucidated requiring Src kinase and potential implication of the C2β isoform of phosphatidylinositol 3 kinase. Our data uncover a novel pathway for β1 integrin–mediated adhesion of importance in cellular processes such as angiogenesis and inflammation.
PMCID: PMC3183016  PMID: 21813734
14.  Recent developments and complexities in neutrophil transmigration 
Current opinion in hematology  2010;17(1):9-17.
Purpose of Review
As the migration of neutrophils from blood to inflamed tissues is an essential component of innate immunity and a key contributing factor to the pathogenesis of inflammatory disorders, this aspect of leukocyte biology continues to be a highly dynamic field of research. This review summarises recent findings in this area, focusing on the mechanisms that mediate neutrophil transmigration, an area where significant progress has been made.
Recent findings
The topics to be covered will include responses that are pre-requisite to neutrophil migration through venular walls such as leukocyte luminal crawling and cellular and molecular changes in leukocytes and endothelial cells (ECs) (eg formation of protrusions) that collectively support leukocyte transendothelial cell migration. Advances in both paracellular and transcellular neutrophil migration through ECs will be discussed, addressing the associated roles and regulation of expression of EC luminal and junctional adhesion molecules. Beyond the endothelium, migration through the vascular pericyte coverage and basement membrane will be reviewed.
The unquestionable role of neutrophils in the development and progression of inflammatory conditions suggests that a better understanding of the tissue- and stimulus-specific mechanisms that mediate this response may identify novel pathways that could be exploited for the development of more specific anti-inflammatory interventions.
PMCID: PMC2882030  PMID: 19864945
transmigration; leukocyte; endothelium; inflammation; transcellular; paracellular
15.  Junctional adhesion molecule (JAM)-C mediates leukocyte infiltration in response to ischemia reperfusion injury 
JAM-C is an adhesion molecule that has multiple roles in inflammation and vascular biology but many aspects of its functions under pathological conditions are unknown. Here we investigated the role of JAM-C in leukocyte migration in response to ischemia reperfusion (I/R) injury.
Methods and Results
Pre-treatment of mice with soluble JAM-C (sJAM-C), used as a pharmacological blocker of JAM-C-mediated reactions, significantly suppressed leukocyte migration in models of kidney and cremaster muscle I/R injury (39 and 51% inhibition, respectively). Furthermore, in the cremaster muscle model (studied by intravital microscopy), both leukocyte adhesion and transmigration were suppressed in JAM-C deficient mice (JAM-C−/−) and enhanced in mice over-expressing JAM-C in their endothelial cells (ECs). Analysis of JAM-C subcellular expression by immunoelectron microscopy indicated that in I/R-injured tissues, EC JAM-C was redistributed from cytoplasmic vesicles and EC junctional sites to non-junctional plasma membranes, a response that may account for the role of JAM-C in both leukocyte adhesion and transmigration under conditions of I/R injury.
The findings demonstrate a role for EC JAM-C in mediating leukocyte adhesion and transmigration in response to I/R injury and indicate the existence of a novel regulatory mechanism for redistribution and hence function of EC JAM-C in vivo.
PMCID: PMC2746810  PMID: 19574560
JAM-C; Ischemia reperfusion injury; Leukocyte transmigration; Inflammation; Adhesion molecules
16.  Monocytes and Neutrophils Exhibit Both Distinct and Common Mechanisms in Penetrating the Vascular Basement Membrane In Vivo 
Leukocyte migration through venular walls is a fundamental event during inflammation, but many aspects of this response, including the mechanisms associated with leukocyte migration through the vascular basement membrane (BM) in vivo, are poorly understood. Here we investigated and compared the means by which neutrophils and monocytes migrate through the venular BM. Specifically, as we have previously reported on the existence of neutrophil permissive sites (termed matrix protein low expression regions; LERs) within the venular BM, we have now investigated the role of these sites in monocyte transmigration in vivo.
Methods and Results
Analysis of CCL2-stimulated mouse cremaster muscles by immunofluorescent staining and confocal microscopy demonstrated that both neutrophils and monocytes use LERs for penetrating venular walls, but independent and distinct mechanisms are used by the 2 cell types. Collectively, (1) neutrophil but not monocyte transmigration led to enlargement of LERs, (2) monocytes showed a greater extent of deformability in migrating through the venular BM, and (3) only extravasated neutrophils were associated with the carriage of laminin fragments.
The findings provide novel insights into mechanisms of leukocyte transmigration by presenting the first in vivo evidence for distinct modes used by neutrophils and monocytes in penetrating the vascular BM.
PMCID: PMC2712455  PMID: 19498176
monocytes; leukocyte transmigration; inflammation; vascular basementmembrane; leukocyte shape-change
17.  Venular basement membranes contain specific matrix protein low expression regions that act as exit points for emigrating neutrophils 
The Journal of Experimental Medicine  2006;203(6):1519-1532.
The mechanism of leukocyte migration through venular walls in vivo is largely unknown. By using immunofluorescence staining and confocal microscopy, the present study demonstrates the existence of regions within the walls of unstimulated murine cremasteric venules where expression of key vascular basement membrane (BM) constituents, laminin 10, collagen IV, and nidogen-2 (but not perlecan) are considerably lower (<60%) than the average expression detected in the same vessel. These sites were closely associated with gaps between pericytes and were preferentially used by migrating neutrophils during their passage through cytokine-stimulated venules. Although neutrophil transmigration did not alter the number/unit area of extracellular matrix protein low expression sites, the size of these regions was enlarged and their protein content was reduced in interleukin-1β–stimulated venules. These effects were entirely dependent on the presence of neutrophils and appeared to involve neutrophil-derived serine proteases. Furthermore, evidence was obtained indicating that transmigrating neutrophils carry laminins on their cell surface in vivo. Collectively, through identification of regions of low extracellular matrix protein localization that define the preferred route for transmigrating neutrophils, we have identified a plausible mechanism by which neutrophils penetrate the vascular BM without causing a gross disruption to its intricate structure.
PMCID: PMC2118318  PMID: 16754715
18.  PECAM-1 (CD31) Homophilic Interaction Up-Regulates α6β1 on Transmigrated Neutrophils In Vivo and Plays a Functional Role in the Ability of α6 Integrins to Mediate Leukocyte Migration through the Perivascular Basement Membrane 
The Journal of Experimental Medicine  2002;196(9):1201-1212.
Platelet-endothelial cell adhesion molecule (PECAM)-1 has been implicated in leukocyte migration through the perivascular basement membrane (PBM) though the mechanisms involved are unclear. The present results demonstrate that the ability of α6 integrins to mediate neutrophil migration through the PBM is PECAM-1 dependent, a response associated with PECAM-1–mediated increased expression of α6β1 on transmigrating neutrophils in vivo. An anti-α6 integrins mAb (GoH3) inhibited (78%, P < 0.001) neutrophil migration through interleukin (IL)-1β–stimulated cremasteric venules, primarily at the level of the PBM, as analyzed by intravital and electron microscopy. In PECAM-1–deficient mice (KO), a reduced level of neutrophil transmigration elicited by IL-1β (4-h reaction) was observed in both the cremaster muscle (55% inhibition, P < 0.05) and in the peritoneum (57% inhibition, P < 0.01) but GoH3 had no additional inhibitory effect on these responses. FACS® analysis of neutrophils demonstrated increased expression of α6β1 on transmigrated peritoneal neutrophils, as compared with blood neutrophils, in wild-type but not KO mice even though neutrophils from both strains of mice exhibited comparable levels of intracellular expression of α6 as observed by immunofluorescent staining and confocal microscopy. Furthermore, mice deficient in either leukocyte or endothelial cell PECAM-1, as developed by bone marrow transplantation, demonstrated a similar level of reduced neutrophil transmigration and expression of α6β1 on transmigrated neutrophils as that detected in KO mice.
The results demonstrate a role for PECAM-1 homophilic interaction in neutrophil transmigration and increased expression of α6β1 on the cell surface of transmigrated neutrophils in vivo, a response that could contribute to the mechanism of PECAM-1–mediated neutrophil migration through the PBM.
PMCID: PMC2194111  PMID: 12417630
inflammation; adhesion molecules; laminin; integrins; intravital microcopy
19.  The peritoneal tumour microenvironment of high-grade serous ovarian cancer 
The Journal of Pathology  2012;227(2):136-145.
High-grade serous ovarian cancer (HGSC) disseminates early and extensively throughout the peritoneal space, causing multiple lesions that are a major clinical problem. The aim of this study was to investigate the cellular composition of peritoneal tumour deposits in patient biopsies and their evolution in mouse models using immunohistochemistry, intravital microscopy, confocal microscopy, and 3D modelling. Tumour deposits from the omentum of HGSC patients contained a prominent leukocyte infiltrate of CD3+ T cells and CD68+ macrophages, with occasional neutrophils. Alpha-smooth muscle actin+ (α-SMA+) pericytes and/or fibroblasts surrounded these well-vascularized tumour deposits. Using the murine bowel mesentery as an accessible mouse peritoneal tissue that could be easily imaged, and two different transplantable models, we found multiple microscopic tumour deposits after i.p. injection of malignant cells. Attachment to the peritoneal surface was rapid (6–48 h) with an extensive CD45+ leukocyte infiltrate visible by 48 h. This infiltrate persisted until end point and in the syngeneic murine ID8 model, it primarily consisted of CD3+ T lymphocytes and CD68+ macrophages with α-SMA+ cells also involved from the earliest stages. A majority of tumour deposits developed above existing mesenteric blood vessels, but in avascular spaces new blood vessels tracked towards the tumour deposits by 2–3 weeks in the IGROV-1 xenografts and 6 weeks in the ID8 syngeneic model; a vigorous convoluted blood supply was established by end point. Inhibition of tumour cell cytokine production by stable expression of shRNA to CXCR4 in IGROV-1 cells did not influence the attachment of cells to the mesentery but delayed neovascularization and reduced tumour deposit size. We conclude that the multiple peritoneal tumour deposits found in HGSC patients can be modelled in the mouse. The techniques described here may be useful for assessing treatments that target the disseminated stage of this disease. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
PMCID: PMC3609073  PMID: 22322968
ovarian cancer; peritoneum; metastases; inflammation; chemokines; CXCR4; intravital microscopy

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