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Granulocyte CEACAM3 is a phagocytic receptor of the innate immune system that mediates recognition and elimination of human-specific pathogens.

Schmitter T, Agerer F, Peterson L, Munzner P, Hauck CR - J. Exp. Med. (2004)

Bottom Line: CEACAM3- but not CEACAM6-mediated uptake is blocked by dominant-negative versions of the small GTPase Rac.Moreover, CEACAM3 engagement triggers membrane recruitment and increased GTP loading of Rac that are not observed upon bacterial binding to CEACAM6.Internalization and Rac stimulation are also inhibited by compromising the integrity of an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 or by interference with Src family protein tyrosine kinases that phosphorylate CEACAM3.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

ABSTRACT
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are used by several human pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that participates together with CEACAM1 and CEACAM6 in the recognition of CEACAM-binding microorganisms. Here we show that CEACAM3 can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella, and Haemophilus species. CEACAM3- but not CEACAM6-mediated uptake is blocked by dominant-negative versions of the small GTPase Rac. Moreover, CEACAM3 engagement triggers membrane recruitment and increased GTP loading of Rac that are not observed upon bacterial binding to CEACAM6. Internalization and Rac stimulation are also inhibited by compromising the integrity of an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 or by interference with Src family protein tyrosine kinases that phosphorylate CEACAM3. In contrast to interfering with CEACAM6, blockage of CEACAM3-mediated events reduces the ability of primary human granulocytes to internalize and eliminate CEACAM-binding bacteria, indicating an important role of CEACAM3 in the control of human-specific pathogens by the innate immune system.

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CEACAM3 engagement by OpaCEA gonococci results in Rac stimulation. (A) 293 cells transfected with the indicated constructs were infected with OpaCEA N. gonorrhoeae for different time points. GTP-bound Rac was isolated using the Rac-binding domain of PAK (GST-CRIB) and detected by Western blotting with anti-Rac antibodies (top). Whole cell lysates (WCL) were also probed with anti-Rac antibodies (bottom). (B) CEACAM3-expressing 293 cells were left uninfected or were infected with N. cinerea, piliated, nonopaque N. gonorrhoeae (Ngo Opa−) or OpaCEA N. gonorrhoeae (Ngo OpaCEA). GST-CRIB pull-downs (top) or whole cell lysates (middle) were probed with anti-Rac antibodies. Lysates of the bacteria were probed with monoclonal anti-Opa antibody (bottom). (C) 293 cells transfected with the empty control vector (pcDNA) or CEACAM3 were infected with the bacteria employed in B and analyzed in gentamicin protection assays. The graph shows mean values ± SDs of three independent experiments done in triplicate.
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fig4: CEACAM3 engagement by OpaCEA gonococci results in Rac stimulation. (A) 293 cells transfected with the indicated constructs were infected with OpaCEA N. gonorrhoeae for different time points. GTP-bound Rac was isolated using the Rac-binding domain of PAK (GST-CRIB) and detected by Western blotting with anti-Rac antibodies (top). Whole cell lysates (WCL) were also probed with anti-Rac antibodies (bottom). (B) CEACAM3-expressing 293 cells were left uninfected or were infected with N. cinerea, piliated, nonopaque N. gonorrhoeae (Ngo Opa−) or OpaCEA N. gonorrhoeae (Ngo OpaCEA). GST-CRIB pull-downs (top) or whole cell lysates (middle) were probed with anti-Rac antibodies. Lysates of the bacteria were probed with monoclonal anti-Opa antibody (bottom). (C) 293 cells transfected with the empty control vector (pcDNA) or CEACAM3 were infected with the bacteria employed in B and analyzed in gentamicin protection assays. The graph shows mean values ± SDs of three independent experiments done in triplicate.

Mentions: Biochemically, Rac stimulation is reflected by its GTP-loading status. Upon infection with OpaCEA gonococci, we observed a strong increase in GTP-Rac in CEACAM3-expressing 293 cells compared with uninfected cells (Fig. 4 A, top). This increase was already detectable 15 min after infection and reached maximum levels between 30 and 60 min paralleling the kinetics of efficient, CEACAM3-mediated internalization. In contrast, no increase in GTP-Rac was observed in control vector-transfected or in CEACAM6-expressing cells after infection (Fig. 4 A, top). An increase in Rac-GTP–loading was only detected in response to OpaCEA-expressing bacteria but not upon infection with nonopaque gonococci or nonpathogenic N. cinerea (Fig. 4 B), demonstrating that it is the specific interaction between the bacterial OpaCEA adhesin and host CEACAM3 that mediates stimulation of Rac. Importantly, the levels of Rac-GTP induced by the different bacterial strains paralleled the internalization of these bacteria via CEACAM3 (Fig. 4 C). Together, these data corroborate the view that CEACAMs might be differentially connected to intracellular signaling pathways and that in particular CEACAM3 might be responsible for the stimulation of the small GTPase Rac in response to OpaCEA gonococci.


Granulocyte CEACAM3 is a phagocytic receptor of the innate immune system that mediates recognition and elimination of human-specific pathogens.

Schmitter T, Agerer F, Peterson L, Munzner P, Hauck CR - J. Exp. Med. (2004)

CEACAM3 engagement by OpaCEA gonococci results in Rac stimulation. (A) 293 cells transfected with the indicated constructs were infected with OpaCEA N. gonorrhoeae for different time points. GTP-bound Rac was isolated using the Rac-binding domain of PAK (GST-CRIB) and detected by Western blotting with anti-Rac antibodies (top). Whole cell lysates (WCL) were also probed with anti-Rac antibodies (bottom). (B) CEACAM3-expressing 293 cells were left uninfected or were infected with N. cinerea, piliated, nonopaque N. gonorrhoeae (Ngo Opa−) or OpaCEA N. gonorrhoeae (Ngo OpaCEA). GST-CRIB pull-downs (top) or whole cell lysates (middle) were probed with anti-Rac antibodies. Lysates of the bacteria were probed with monoclonal anti-Opa antibody (bottom). (C) 293 cells transfected with the empty control vector (pcDNA) or CEACAM3 were infected with the bacteria employed in B and analyzed in gentamicin protection assays. The graph shows mean values ± SDs of three independent experiments done in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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fig4: CEACAM3 engagement by OpaCEA gonococci results in Rac stimulation. (A) 293 cells transfected with the indicated constructs were infected with OpaCEA N. gonorrhoeae for different time points. GTP-bound Rac was isolated using the Rac-binding domain of PAK (GST-CRIB) and detected by Western blotting with anti-Rac antibodies (top). Whole cell lysates (WCL) were also probed with anti-Rac antibodies (bottom). (B) CEACAM3-expressing 293 cells were left uninfected or were infected with N. cinerea, piliated, nonopaque N. gonorrhoeae (Ngo Opa−) or OpaCEA N. gonorrhoeae (Ngo OpaCEA). GST-CRIB pull-downs (top) or whole cell lysates (middle) were probed with anti-Rac antibodies. Lysates of the bacteria were probed with monoclonal anti-Opa antibody (bottom). (C) 293 cells transfected with the empty control vector (pcDNA) or CEACAM3 were infected with the bacteria employed in B and analyzed in gentamicin protection assays. The graph shows mean values ± SDs of three independent experiments done in triplicate.
Mentions: Biochemically, Rac stimulation is reflected by its GTP-loading status. Upon infection with OpaCEA gonococci, we observed a strong increase in GTP-Rac in CEACAM3-expressing 293 cells compared with uninfected cells (Fig. 4 A, top). This increase was already detectable 15 min after infection and reached maximum levels between 30 and 60 min paralleling the kinetics of efficient, CEACAM3-mediated internalization. In contrast, no increase in GTP-Rac was observed in control vector-transfected or in CEACAM6-expressing cells after infection (Fig. 4 A, top). An increase in Rac-GTP–loading was only detected in response to OpaCEA-expressing bacteria but not upon infection with nonopaque gonococci or nonpathogenic N. cinerea (Fig. 4 B), demonstrating that it is the specific interaction between the bacterial OpaCEA adhesin and host CEACAM3 that mediates stimulation of Rac. Importantly, the levels of Rac-GTP induced by the different bacterial strains paralleled the internalization of these bacteria via CEACAM3 (Fig. 4 C). Together, these data corroborate the view that CEACAMs might be differentially connected to intracellular signaling pathways and that in particular CEACAM3 might be responsible for the stimulation of the small GTPase Rac in response to OpaCEA gonococci.

Bottom Line: CEACAM3- but not CEACAM6-mediated uptake is blocked by dominant-negative versions of the small GTPase Rac.Moreover, CEACAM3 engagement triggers membrane recruitment and increased GTP loading of Rac that are not observed upon bacterial binding to CEACAM6.Internalization and Rac stimulation are also inhibited by compromising the integrity of an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 or by interference with Src family protein tyrosine kinases that phosphorylate CEACAM3.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

ABSTRACT
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are used by several human pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that participates together with CEACAM1 and CEACAM6 in the recognition of CEACAM-binding microorganisms. Here we show that CEACAM3 can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella, and Haemophilus species. CEACAM3- but not CEACAM6-mediated uptake is blocked by dominant-negative versions of the small GTPase Rac. Moreover, CEACAM3 engagement triggers membrane recruitment and increased GTP loading of Rac that are not observed upon bacterial binding to CEACAM6. Internalization and Rac stimulation are also inhibited by compromising the integrity of an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 or by interference with Src family protein tyrosine kinases that phosphorylate CEACAM3. In contrast to interfering with CEACAM6, blockage of CEACAM3-mediated events reduces the ability of primary human granulocytes to internalize and eliminate CEACAM-binding bacteria, indicating an important role of CEACAM3 in the control of human-specific pathogens by the innate immune system.

Show MeSH
Related in: MedlinePlus