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Leukocyte responses to pathogens: integrins, membrane rafts and nitric oxide
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During microbial invasion, leukocytes of the innate immunity are rapidly recruited to the site of infection where they internalize (phagocytose), kill and digest the invaders. To aid this process, leukocytes express surface receptors such as Toll-like receptors, β2-integrins and Fc-receptors. The β2-integrins are also used for attachment to the extracellular matrix and are important for migration. When pro- vs. anti-inflammatory regulation of β2-integrins was investigated, it was found that chemotactic factors modulate neutrophil adhesion through altered affinity and/or avidity of β2-integrins. A bacteria-derived chemoattractant evoked a large increase in affinity as well as in mobility and clustering, while an early, host-derived chemotactic factor induced increased clustering and surface mobility, but only a slight increase in affinity. Anti-inflammatory lipoxin affected β2-integrin avidity, but not affinity.

The leukocyte membrane is composed of lipids and proteins, which are inhomogeneously distributed. Specific domains in the membrane, membrane rafts, are enriched in signaling proteins and receptors. It was found that lipophosphoglycan (LPG) a virulence factor and membrane component of the parasite Leishmania donovani, accumulated in macrophage rafts during infection, inhibited PKCα translocation to the membrane and halted phagosomal maturation. Membrane rafts were instrumental for LPG to exert its effect. We further showed that nitric oxide (NO) rescued phagosomal maturation halted by Leishmania donovani parasites, possibly through effects on actin dynamics. NO did not affect parasite virulence per se. Moreover, lipoarabinomannan (LAM), a virulence factor on Mycobacterium tuberculosis (Mtb) bacteria, also inserted itself into macrophage membrane rafts. LAM from a less virulent strain (PILAM) was less efficiently inserted. Insertion could to some extent be inhibited by phosphatidylinositol mannoside (PIM), another structural molecule from Mtb. LAM did not activate the p38 MAPK signaling pathway nor did LAM interfere with TLR 2 or 4 signaling. In neutrophil leukocytes we observed a simultaneous, calciumdependent up-regulation of membrane rafts and secretion of azurophilic granules at the site of phagocytosis. Rafts were also found in the phagosome membrane. Wild type Streptococcus pyogenes bacteria, which can survive phagocytosis, modulated raft delivery.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2008. , 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1058
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:liu:diva-43383Local ID: 73683ISBN: 978-91-7393-921-8 (print)OAI: oai:DiVA.org:liu-43383DiVA: diva2:264242
Public defence
2008-05-28, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2015-11-19Bibliographically approved
List of papers
1. Differential inside-out activation of β2 integrins by leukotriene B4 and fMLP in human neutrophils
Open this publication in new window or tab >>Differential inside-out activation of β2 integrins by leukotriene B4 and fMLP in human neutrophils
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2004 (English)In: Experimental Cell Research, ISSN 0014-4827, Vol. 300, no 2, 308-319 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated how LTB4, an endogenous chemoattractant encountered early in the inflammatory process, and fMLP, a bacteria-derived chemotactic peptide emanating from the site of infection, mediate inside-out regulation of the β2-integrin. The role of the two chemoattractants on β2-integrin avidity was investigated by measuring their effect on β2-integrin clustering and surface mobility, whereas their effect on β2-integrin affinity was measured by the expression of a high affinity epitope, a ligand-binding domain on β2-integrins, and by integrin binding to s-ICAM. We find that the two chemoattractants modulate the β2-integrin differently. LTB4 induces an increase in integrin clustering and surface mobility, but only a modest increase in integrin affinity. fMLP evokes a large increase in β2-integrin affinity as well as in clustering and mobility. Lipoxin, which acts as a stop signal for the functions mediated by pro-inflammatory agents, was used as a tool for further examining the inside-out mechanisms. While LTB4-induced integrin clustering and mobility were inhibited by lipoxin, only a minor inhibition of fMLP-induced β2-integrin avidity and no inhibition of integrin affinity were detected. The different modes of the inside-out regulation of β2-integrins suggest that distinct mechanisms are involved in the β2-integrin modulation induced by various chemoattractants.

Keyword
β2-Integrins, Cell adhesion, Chemotactic factors, Eicosanoids, Inflammation, Leukotriene B4, Lipoxins, Human Neutrophils, Signal transduction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14629 (URN)10.1016/j.yexcr.2004.07.015 (DOI)
Available from: 2007-09-13 Created: 2007-09-13 Last updated: 2009-12-14Bibliographically approved
2. Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts
Open this publication in new window or tab >>Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts
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2009 (English)In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 11, no 2, 215-222 p.Article in journal (Refereed) Published
Abstract [en]

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Gal beta 1,4Man alpha-PO4 attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.

Keyword
Leishmania, Lipophosphoglycan, Membrane rafts, Phagosomal maturation, Actin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17886 (URN)10.1016/j.micinf.2008.11.007 (DOI)
Note

Original Publication: Martin Winberg Tinnerfelt, Åsa Holm, Eva Särndahl, Adrien F Vinet, Albert Descoteaux, Karl-Eric Magnusson, Birgitta Rasmusson and Maria Lerm, Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts, 2009, MICROBES AND INFECTION, (11), 2, 215-222. http://dx.doi.org/10.1016/j.micinf.2008.11.007 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/

Available from: 2009-05-20 Created: 2009-04-24 Last updated: 2014-01-13Bibliographically approved
3. Leishmania donovani: Inhibition of phagosomal maturation is rescued by nitric oxide in macrophages
Open this publication in new window or tab >>Leishmania donovani: Inhibition of phagosomal maturation is rescued by nitric oxide in macrophages
2007 (English)In: Experimental parasitology, ISSN 0014-4894, Vol. 117, no 2, 165-170 p.Article in journal (Refereed) Published
Abstract [en]

Leishmania donovani promastigotes, the causative agent of visceral leishmaniasis, survive inside macrophages by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for parasite survival. LPG has several detrimental effects on macrophage function, including inhibition of periphagosomal filamentous actin (F-actin) breakdown during phagosomal maturation. However, in RAW 264.7 macrophages pre-stimulated with lipopolysaccharide (LPS) and interferon ? (IFN?), known to up-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, L. donovani promastigotes are unable to inhibit periphagosomal F-actin breakdown and phagosomal maturation proceeds normally. Moreover, the iNOS inhibitor aminoguanidine, blocked the positive effects of LPS/IFN? suggesting that NO is a key player in F-actin remodeling. In conclusion, production of NO by stimulated macrophages seems to allow phagosomal maturation following uptake of L. donovani promastigotes, suggesting a novel mechanism whereby NO facilitates killing of an intracellular pathogen. © 2007 Elsevier Inc. All rights reserved.

Keyword
Actin, AG, aminoguanidine, bovine serum albumine, BSA, cGMP, F-actin, filamentous actin, gamma Interferon, GFP, green fluorescent protein, guanosine 3':5'-cyclic monophosphate, IFN?, inducible nitric oxide synthase, iNOS, Leishmania donovani, lipophosphoglycan, lipopolysaccharide, LPG, LPS, Maturation, Nitric oxide, nitric oxide, NO, paraformaldehyde, PFA, Phagosome
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-48433 (URN)10.1016/j.exppara.2007.04.004 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2009-12-14Bibliographically approved
4. Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
Open this publication in new window or tab >>Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.
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2008 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 76, no 7, 2882-2887 p.Article in journal (Refereed) Published
Abstract [en]

Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-20816 (URN)10.1128/IAI.01549-07 (DOI)18426888 (PubMedID)
Available from: 2009-09-22 Created: 2009-09-22 Last updated: 2011-02-22Bibliographically approved
5. Phagosomal membrane rafts: azurophilic origin, Ca2+ dependence, and modulation by Streptococcus pyogenes bacteria
Open this publication in new window or tab >>Phagosomal membrane rafts: azurophilic origin, Ca2+ dependence, and modulation by Streptococcus pyogenes bacteria
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Uptake and killing of microorganisms by neutrophils involve tightly regulated membrane traffic events that are governed by complex signals. Many of these are raft-associated, which implies that raft dynamics may be important during phagosome formation. Locally restricted, calcium-dependent, parallel upregulation of markers for membrane rafts and azurophilic granules was observed at the site of phagocytosis of IgG-opsonized prey in human neutrophils. Subsequent internalization of the prey reduced the levels of these markers in the plasma membrane. Streptococcus pyogenes bacteria, that can survive phagocytosis by neutrophils, modulated phagosomal raft acquisition by means of M proteins. Continued, but not early, delivery of rafts to the membrane of phagosomes in neutrophils and HL-60 cells was independent of calcium, as was fusion between azurophilic granules and phagosomes. Nevertheless, calcium depletion affected bacterial killing kinetics. These findings suggest that early delivery of membrane rafts is important for phagosomal maturation in neutrophils and provide new mechanistic insight into the processes required for generation of bactericidal phagosomes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-52261 (URN)
Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2009-12-14Bibliographically approved

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