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A close-up on neutrophils: Visualizing the mechanisms of their in vivo recruitment and function
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology. (Mia Phillipson)ORCID iD: 0000-0003-2232-2871
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies.

The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice.

In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 56 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1159
Keyword [en]
angiogenesis, chemokine, chemotactic gradient, citrullination, hypoxia, inflammation, intraluminal crawling, intravital imaging, PAD enzymes, permeability, proangiogenic, proinflammatory
National Category
URN: urn:nbn:se:uu:diva-265203ISBN: 978-91-554-9401-8OAI: oai:DiVA.org:uu-265203DiVA: diva2:866164
Public defence
2015-12-18, A1:107a, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2015-11-26 Created: 2015-10-25 Last updated: 2016-01-13
List of papers
1. A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils
Open this publication in new window or tab >>A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils
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2010 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 116, no 11, 1924-1931 p.Article in journal (Refereed) Published
Abstract [en]

During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 super-fusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site.

National Category
Medical and Health Sciences
urn:nbn:se:uu:diva-134806 (URN)10.1182/blood-2010-01-266072 (DOI)000282152000018 ()20530797 (PubMedID)
Available from: 2010-12-01 Created: 2010-12-01 Last updated: 2016-01-13Bibliographically approved
2. Increased vascular permeability facilitates vascular influx of chemokines and accelerates recruitment of circulating neutrophils
Open this publication in new window or tab >>Increased vascular permeability facilitates vascular influx of chemokines and accelerates recruitment of circulating neutrophils
(English)Manuscript (preprint) (Other academic)
National Category
urn:nbn:se:uu:diva-265202 (URN)
Available from: 2015-11-01 Created: 2015-10-25 Last updated: 2016-01-13
3. Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans
Open this publication in new window or tab >>Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans
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2015 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 126, no 17, 2016-2026 p.Article in journal (Refereed) Published
Abstract [en]

Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

National Category
urn:nbn:se:uu:diva-265201 (URN)10.1182/blood-2015-03-631572 (DOI)000366389200012 ()26286848 (PubMedID)
Swedish Research CouncilThe Royal Swedish Academy of SciencesMagnus Bergvall FoundationSwedish Diabetes AssociationÅke Wiberg FoundationRagnar Söderbergs stiftelseKnut and Alice Wallenberg Foundation
Available from: 2015-10-25 Created: 2015-10-25 Last updated: 2016-08-26Bibliographically approved

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