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Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7.

Verbovetski I, Bychkov H, Trahtemberg U, Shapira I, Hareuveni M, Ben-Tal O, Kutikov I, Gill O, Mevorach D - J. Exp. Med. (2002)

Bottom Line: A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios.In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited.We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.

View Article: PubMed Central - PubMed

Affiliation: The Laboratory for Cellular and Molecular Immunology, Rheumatology Unit, Department of Medicine, Hadassah Hospital and the Hebrew University. Sourasky Medical Center, Jerusalem 91120, Israel.

ABSTRACT
Immature dendritic cells (iDCs) do not mature after uptake of apoptotic cells and may play a role in the induction of peripheral tolerance to self antigens derived from apoptotic material. The integrins, alphavbeta3, alphavbeta5, and the scavenger receptor, CD36, have been shown to mediate uptake of apoptotic cells by iDCs. However, it is not known whether the complement system, also takes part in this process. In this study we investigated the ability of iDCs to bind to apoptotic cells opsonized by iC3b. Monocyte-derived dendritic cells were offered apoptotic Jurkat cells opsonized by autologous iC3b and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate. A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios. Despite increased efficiency of uptake, interaction between iC3b-opsonized apoptotic cells and iDCs down-regulated the expression of major histocompatibility complex class II, CD86, CC chemokine receptor (CCR)2, CCR5, and beta2-integrins (P < 0.001), and up-regulated expression of CCR7 (P < 0.001). In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited. We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.

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Apoptotic Jurkat cells are opsonized by iC3b. Jurkat cells were induced to undergo serum withdrawal apoptosis, followed by incubation and exposure to 15% fresh serum from donor dendritic cells for 1 h/37°C/5% CO2. Cells were stained with annexin V-FITC (A), or iC3b-PE (B). As seen by confocal microscopy (Nomarsky, 40, zoom ×2), cells show membranous staining of both Phosphatidylserine and iC3b. Viable cells or apoptotic cells exposed to heat-inactivated serum did not stain for iC3b.
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fig1: Apoptotic Jurkat cells are opsonized by iC3b. Jurkat cells were induced to undergo serum withdrawal apoptosis, followed by incubation and exposure to 15% fresh serum from donor dendritic cells for 1 h/37°C/5% CO2. Cells were stained with annexin V-FITC (A), or iC3b-PE (B). As seen by confocal microscopy (Nomarsky, 40, zoom ×2), cells show membranous staining of both Phosphatidylserine and iC3b. Viable cells or apoptotic cells exposed to heat-inactivated serum did not stain for iC3b.

Mentions: The conditions for serum-withdrawal apoptosis were selected because 60–70% of cells were annexin-V positive and <7% were positive for propidium iodide staining (unpublished data). Activation of complement by apoptotic Jurkat cells was shown previously by us and by others (9, 15, 16). However, in order to verify that apoptotic Jurkat cells can activate complement under the specific conditions that we used for induction of apoptosis, we measured release of C3a in the supernatants. C3a release measured in the supernatants of interacting iDCs and apoptotic Jurkat cells was found to be 1,990 ±198 ng/ml in the presence of 15% fresh serum as compared with 220 ± 38 when the interaction occurred in the presence of heat-inactivated serum (P < 0.001). To further verify that apoptotic cells were opsonized by iC3b we stained apoptotic Jurkat cells for iC3b deposition and found membrane staining using confocal microscopy (Fig. 1) . These results were verified using flow cytometry, where deposition of iC3b was shown in >90% of annexin-V–positive cells but in <3% of annexin-V–negative cells (not shown).


Opsonization of apoptotic cells by autologous iC3b facilitates clearance by immature dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7.

Verbovetski I, Bychkov H, Trahtemberg U, Shapira I, Hareuveni M, Ben-Tal O, Kutikov I, Gill O, Mevorach D - J. Exp. Med. (2002)

Apoptotic Jurkat cells are opsonized by iC3b. Jurkat cells were induced to undergo serum withdrawal apoptosis, followed by incubation and exposure to 15% fresh serum from donor dendritic cells for 1 h/37°C/5% CO2. Cells were stained with annexin V-FITC (A), or iC3b-PE (B). As seen by confocal microscopy (Nomarsky, 40, zoom ×2), cells show membranous staining of both Phosphatidylserine and iC3b. Viable cells or apoptotic cells exposed to heat-inactivated serum did not stain for iC3b.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196062&req=5

fig1: Apoptotic Jurkat cells are opsonized by iC3b. Jurkat cells were induced to undergo serum withdrawal apoptosis, followed by incubation and exposure to 15% fresh serum from donor dendritic cells for 1 h/37°C/5% CO2. Cells were stained with annexin V-FITC (A), or iC3b-PE (B). As seen by confocal microscopy (Nomarsky, 40, zoom ×2), cells show membranous staining of both Phosphatidylserine and iC3b. Viable cells or apoptotic cells exposed to heat-inactivated serum did not stain for iC3b.
Mentions: The conditions for serum-withdrawal apoptosis were selected because 60–70% of cells were annexin-V positive and <7% were positive for propidium iodide staining (unpublished data). Activation of complement by apoptotic Jurkat cells was shown previously by us and by others (9, 15, 16). However, in order to verify that apoptotic Jurkat cells can activate complement under the specific conditions that we used for induction of apoptosis, we measured release of C3a in the supernatants. C3a release measured in the supernatants of interacting iDCs and apoptotic Jurkat cells was found to be 1,990 ±198 ng/ml in the presence of 15% fresh serum as compared with 220 ± 38 when the interaction occurred in the presence of heat-inactivated serum (P < 0.001). To further verify that apoptotic cells were opsonized by iC3b we stained apoptotic Jurkat cells for iC3b deposition and found membrane staining using confocal microscopy (Fig. 1) . These results were verified using flow cytometry, where deposition of iC3b was shown in >90% of annexin-V–positive cells but in <3% of annexin-V–negative cells (not shown).

Bottom Line: A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios.In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited.We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.

View Article: PubMed Central - PubMed

Affiliation: The Laboratory for Cellular and Molecular Immunology, Rheumatology Unit, Department of Medicine, Hadassah Hospital and the Hebrew University. Sourasky Medical Center, Jerusalem 91120, Israel.

ABSTRACT
Immature dendritic cells (iDCs) do not mature after uptake of apoptotic cells and may play a role in the induction of peripheral tolerance to self antigens derived from apoptotic material. The integrins, alphavbeta3, alphavbeta5, and the scavenger receptor, CD36, have been shown to mediate uptake of apoptotic cells by iDCs. However, it is not known whether the complement system, also takes part in this process. In this study we investigated the ability of iDCs to bind to apoptotic cells opsonized by iC3b. Monocyte-derived dendritic cells were offered apoptotic Jurkat cells opsonized by autologous iC3b and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate. A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios. Despite increased efficiency of uptake, interaction between iC3b-opsonized apoptotic cells and iDCs down-regulated the expression of major histocompatibility complex class II, CD86, CC chemokine receptor (CCR)2, CCR5, and beta2-integrins (P < 0.001), and up-regulated expression of CCR7 (P < 0.001). In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited. We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes.

Show MeSH
Related in: MedlinePlus