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Signal transduction in human phagocytic cells during phagocytosis, oxidative activation and apoptosis
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neutrophils and macrophages are professional phagocytic cells that play a crucial role in host defense against invading microorganisms. They bind to, internalize, and subsequently kill microbes with an arsenal of reactive oxygen metabolites and microbicidal agents. The microbes are recognized by cell surface receptors, mainly by the phagocytic receptors FcγR and complement receptor 3 (CR3) that recognize IgG and complement fragments C3b/C3bi, respectively. Microbial pathogens such as Salmonella typhimurium have developed sophisticated mechanisms to avoid the host defense system and enter the cells by invasion, mediated by a type III secretion system.

The objective of this thesis was to investigate the signaling pathways during receptor-mediated phagocytosis by FcγRIIa, FcγRIIIb and complement receptor 3 (CR3), or during invasion by Salmonella typhimurium in human phagocytic cells. We have focused on the intracellular signaling pathways controlling phagocytosis, production of reactive oxygen metabolites, and apoptosis. Paper I-III focus on signal transduction events triggered after ligation of CR3, FcγRIIa, and FcγRIIIb in human neutrophils. Both activation of CR3 and FcγR induced production of reactive oxygen metabolites (ROM), where CR3 induced the most prominent response. The ROM production was dependent on intracellular Ca2+, tyrosine kinase activation, and phospholipase D (PLD) activity. FcγRIIa induced a strong phosphorylation Syk, which was less pronounced following FcγRIIIb ligation, and absent after CR3 activation. Our data indicate that CR3 and FcγR activate different signaling pathways. By exposing neutrophils to TNF-α prior to ligation of CR3, the oxidative response was strongly enhanced, whereas the response to FcγR-ligation was unaffected. This increase was in part due to a p38 MAPK-dependent upregulation of CR3 on the cell surface, but also due to modulation of intracellular signaling pathways since Syk was activated by CR3 as well as FcγR in TNF-α treated cells. In contrast to macrophages where only FcγR activates Rac, Cdc42, and the subsequent ROM production, we show that CR3 as well as FcγR activate the GTPases Rac2 and Cdc42 in human neutrophils. Their downstream target p21 activated kinase was also activated, and Rac2 translocated to the membrane fraction. Correct function of these small GTP-binding proteins was necessary for generating a proper signal for ROM production in these cells.

One survival strategy exploited by microbial pathogens might be to induce apoptosis of tbe host. Invasive Salmonella typhimurium efficiently entered U937 cells and induced a pronounced degree of apoptosis in contrast to its opsonized mutants, which were internalized by receptor-mediated phagocytosis but failed to induce apoptosis. Invasion by Salmonella typhimurium activated Rac1 and Cdc42 independently of PI3 K and tyrosine kinase activation. Inhibition of Racl and Cdc42 inhibited both invasion and the induction of apoptosis. Receptor-mediated phagocytosis activated the survival signals Akt/PKB which protected the cells from apoptosis. Thus, control of apoptosis is a fine tuned balance between pro- and anti-apoptotic signaling proteins.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 789
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26651Local ID: 11216ISBN: 91-7373-548-5 (print)OAI: oai:DiVA.org:liu-26651DiVA: diva2:247200
Public defence
2003-05-09, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-09Bibliographically approved
List of papers
1. CR3, FcγRIIA and FcγRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca2+, phospholipase D and tyrosine phosphorylation
Open this publication in new window or tab >>CR3, FcγRIIA and FcγRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca2+, phospholipase D and tyrosine phosphorylation
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1999 (English)In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, Vol. 1452, no 1, 46-59 p.Article in journal (Refereed) Published
Abstract [en]

Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcγRIIA and FcγRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcγRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcγRIIA Pansorbins induced an even weaker signal. However, FcγRIIA induced strong phosphorylation of p72syk whereas FcγRIIIB induced only a very weak p72syk phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72syk was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72syk that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72syk phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcγRIIA and FcγRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72syk is an early signal in the cascade induced by FcγRIIA but not by CR3.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25448 (URN)10.1016/S0167-4889(99)00112-3 (DOI)9894 (Local ID)9894 (Archive number)9894 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-09Bibliographically approved
2. Tumour necrosis factor-α potentiates CR3-induced respiratory burst by activating p38 MAP kinase in human neutrophils
Open this publication in new window or tab >>Tumour necrosis factor-α potentiates CR3-induced respiratory burst by activating p38 MAP kinase in human neutrophils
2001 (English)In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 103, no 4, 465-472 p.Article in journal (Refereed) Published
Abstract [en]

CR3 and FcγRs are the main receptors involved in the phagocytic process leading to engulfment and killing of microbes by production of reactive oxygen intermediates (ROI) and degranulation. Various inflammatory mediators, such as tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), are known to prime neutrophils leading to increased bactericidal responses, but the underlying mechanism of priming has only been partially elucidated. The purpose of this study was to investigate how TNF-α primes neutrophils for subsequent stimuli via either CR3 or FcγR. The receptors were specifically activated with pansorbins (protein-A-positive Staphylococcus aureus) coated with anti-CR3, anti-FcγRIIa, or anti-FcγRIIIb monoclonal antibody. Activation of neutrophils with these particles resulted in ROI production as measured by chemiluminescence. Anti-CR3 pansorbins induced the most prominent ROI production in neutrophils. TNF-α potentiated the CR3-mediated respiratory burst but had little effect on that mediated by FcγRs. The priming effect of TNF-α on CR3-mediated ROI production is associated with an increased activation of p38 MAPK as well as tyrosine phosphorylation of p72syk. Pretreatment of neutrophils with the inhibitors for p38 MAPK and p72syk markedly suppressed the respiratory burst induced by CR3. Furthermore, TNF-α induced about a three-fold increase in the expression of CR3 in neutrophils, an effect which is blocked by the p38 MAPK inhibitor. Taken together, these results showed that TNF-α potentiates the CR3-mediated respiratory burst in neutrophils not only by triggering a p38 MAPK-dependent up-regulation of CD11b/CD18 but also by modulating the signalling pathways.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25910 (URN)10.1046/j.1365-2567.2001.01270.x (DOI)10352 (Local ID)10352 (Archive number)10352 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-09Bibliographically approved
3. Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils
Open this publication in new window or tab >>Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils
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2003 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, 611-619 p.Article in journal (Refereed) Published
Abstract [en]

Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcγR)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcγR and CR3- and FcγR-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or FcγRs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24991 (URN)10.1189/jlb.1102525 (DOI)9411 (Local ID)9411 (Archive number)9411 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-09Bibliographically approved
4. Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt
Open this publication in new window or tab >>Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt
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2003 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, 620-629 p.Article in journal (Refereed) Published
Abstract [en]

In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.

Keyword
macrophages, bacterial apoptosis, signal transduction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14003 (URN)10.1189/jlb.1202586 (DOI)
Available from: 2006-09-27 Created: 2006-09-27 Last updated: 2012-10-09Bibliographically approved

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