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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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Evaluation of interaction between iDCs and apoptotic Jurkat cells using flow cytometry. (Left) Jurkat cells were stained with DIL and then were induced to undergo apoptosis. DIL-stained apoptotic cells (bold line) and unstained apoptotic cells (solid line) are shown. (Middle) When iDCs and Jurkat cells were incubated together at 4°C, immediately before flow cytometry acquisition, two subpopulations were recognized. R1 consisted of >93% CD1a+CD3−DIL− cells, whereas R2 shows >94% CD1a−CD3+DIL+ cells. Thus, R1 determined population of noninteracting iDCs and R2 determined population of unbound Jurkat cells. During a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells at 37°C, iDCs, acquired DIL from bound or phagocytosed apoptotic cells. Thus, after interaction at 37°C, R1 contains both noninteracting iDCs and interacting iDCs. (Right) R1 of the former figure after a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells, at 37°C. Most of the interacting iDCs (bold line) acquired DIL in this experiment as compared with unstained iDCs (dotted line). iDCs that did not bound or phagocytosed apoptotic cells are found in the area of unstained iDCs. During 2 h of interaction no passive staining of iDCs occurred as demonstrated by 2 chambers incubation (not shown). The percentage of interacting iDCs that acquired DIL, as well as, mean and median fluorescence, were measured. The interaction was performed in different iDC:Jurkat ratios and in the presence or absence of fresh serum or heat inactivated serum. In inhibition studies, iDCs were preincubated with monoclonal antibodies for 30 min before interaction. To distinguish between bound apoptotic Jurkat cells and internalized cells, we stained with anti-CD3-FITC and determine what percentage of iDCs that acquired DIL, stained for CD3. CD3, expressed on apoptotic Jurkat cells (and not on iDCs), stained only noninternalized, adhered, apoptotic Jurkat cells. Similar results were obtained upon validation of this system, using Jurkat cells stained with PKH, or double staining of CD1a-FITC, DIL-apoptotic Jurkat cells.
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fig2: Evaluation of interaction between iDCs and apoptotic Jurkat cells using flow cytometry. (Left) Jurkat cells were stained with DIL and then were induced to undergo apoptosis. DIL-stained apoptotic cells (bold line) and unstained apoptotic cells (solid line) are shown. (Middle) When iDCs and Jurkat cells were incubated together at 4°C, immediately before flow cytometry acquisition, two subpopulations were recognized. R1 consisted of >93% CD1a+CD3−DIL− cells, whereas R2 shows >94% CD1a−CD3+DIL+ cells. Thus, R1 determined population of noninteracting iDCs and R2 determined population of unbound Jurkat cells. During a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells at 37°C, iDCs, acquired DIL from bound or phagocytosed apoptotic cells. Thus, after interaction at 37°C, R1 contains both noninteracting iDCs and interacting iDCs. (Right) R1 of the former figure after a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells, at 37°C. Most of the interacting iDCs (bold line) acquired DIL in this experiment as compared with unstained iDCs (dotted line). iDCs that did not bound or phagocytosed apoptotic cells are found in the area of unstained iDCs. During 2 h of interaction no passive staining of iDCs occurred as demonstrated by 2 chambers incubation (not shown). The percentage of interacting iDCs that acquired DIL, as well as, mean and median fluorescence, were measured. The interaction was performed in different iDC:Jurkat ratios and in the presence or absence of fresh serum or heat inactivated serum. In inhibition studies, iDCs were preincubated with monoclonal antibodies for 30 min before interaction. To distinguish between bound apoptotic Jurkat cells and internalized cells, we stained with anti-CD3-FITC and determine what percentage of iDCs that acquired DIL, stained for CD3. CD3, expressed on apoptotic Jurkat cells (and not on iDCs), stained only noninternalized, adhered, apoptotic Jurkat cells. Similar results were obtained upon validation of this system, using Jurkat cells stained with PKH, or double staining of CD1a-FITC, DIL-apoptotic Jurkat cells.

Mentions: We generated iDCs that were >90% CD14−CD1a+, as well as low DR, CD83, and CD86 (see below). To determine whether complement activation increased uptake of apoptotic cells, we measured uptake by iDCs by FACScan™ using similar methodology to that described (4, 7, 17), and as explained in Fig. 2 . As shown in Fig. 3 A, at a ratio of 1:1, iDC:apoptotic cell, 44.25 ± 2.21% of iDCs bound/engulfed apoptotic Jurkat cells in the absence of serum compared with 83.25 ± 4.3%, in the presence of serum (P < 0.001, triplicates, representative of six experiments). At a ratio of 1:2 the gap decreased but there was still a significant difference in all experiments, 59.2 ± 4.3% versus 93 ± 5.3%, respectively (P < 0.001). However, at 1:4 there was a further decrease and the difference was nonsignificant in one out of six experiments. At ratios of 1:8 and 1:16 there was no difference in the number of iDCs that bound/engulfed apoptotic Jurkat cells in the absence or presence of autologous fresh serum. To further evaluate the relative number of apoptotic cells bound or engulfed by iDCs, we used the median fluorescence of DIL detected in the area of the gated iDCs. As shown in Fig. 3 B, median fluorescence, at a ratio of 1:1, was increased up to eightfold in the presence of autologous serum. This difference decreased gradually upon increasing the ratio but was still elevated (1.3–1.8-fold) at a ratio of 1:16. To further verify that this effect was due to complement activation we simultaneously examined interactions with autologous heat-inactivated serum. Heat-inactivation abolished the effect of serum, the result being similar to that seen in the absence of serum (Fig. 3, C and D). The experiments were repeated using autologous plasma instead of serum, with similar results (not shown). To verify that all interacting cells were phagocytosed, staining with anti-CD3 was performed. 2 h after interaction, between 18–35% of the interacting cells still had bound Jurkat cells (at ratio 1:4). However, by 18 h after the interaction, <10% of iDCs that acquired DIL, stained for CD3.


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)

Evaluation of interaction between iDCs and apoptotic Jurkat cells using flow cytometry. (Left) Jurkat cells were stained with DIL and then were induced to undergo apoptosis. DIL-stained apoptotic cells (bold line) and unstained apoptotic cells (solid line) are shown. (Middle) When iDCs and Jurkat cells were incubated together at 4°C, immediately before flow cytometry acquisition, two subpopulations were recognized. R1 consisted of >93% CD1a+CD3−DIL− cells, whereas R2 shows >94% CD1a−CD3+DIL+ cells. Thus, R1 determined population of noninteracting iDCs and R2 determined population of unbound Jurkat cells. During a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells at 37°C, iDCs, acquired DIL from bound or phagocytosed apoptotic cells. Thus, after interaction at 37°C, R1 contains both noninteracting iDCs and interacting iDCs. (Right) R1 of the former figure after a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells, at 37°C. Most of the interacting iDCs (bold line) acquired DIL in this experiment as compared with unstained iDCs (dotted line). iDCs that did not bound or phagocytosed apoptotic cells are found in the area of unstained iDCs. During 2 h of interaction no passive staining of iDCs occurred as demonstrated by 2 chambers incubation (not shown). The percentage of interacting iDCs that acquired DIL, as well as, mean and median fluorescence, were measured. The interaction was performed in different iDC:Jurkat ratios and in the presence or absence of fresh serum or heat inactivated serum. In inhibition studies, iDCs were preincubated with monoclonal antibodies for 30 min before interaction. To distinguish between bound apoptotic Jurkat cells and internalized cells, we stained with anti-CD3-FITC and determine what percentage of iDCs that acquired DIL, stained for CD3. CD3, expressed on apoptotic Jurkat cells (and not on iDCs), stained only noninternalized, adhered, apoptotic Jurkat cells. Similar results were obtained upon validation of this system, using Jurkat cells stained with PKH, or double staining of CD1a-FITC, DIL-apoptotic Jurkat cells.
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Related In: Results  -  Collection

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

fig2: Evaluation of interaction between iDCs and apoptotic Jurkat cells using flow cytometry. (Left) Jurkat cells were stained with DIL and then were induced to undergo apoptosis. DIL-stained apoptotic cells (bold line) and unstained apoptotic cells (solid line) are shown. (Middle) When iDCs and Jurkat cells were incubated together at 4°C, immediately before flow cytometry acquisition, two subpopulations were recognized. R1 consisted of >93% CD1a+CD3−DIL− cells, whereas R2 shows >94% CD1a−CD3+DIL+ cells. Thus, R1 determined population of noninteracting iDCs and R2 determined population of unbound Jurkat cells. During a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells at 37°C, iDCs, acquired DIL from bound or phagocytosed apoptotic cells. Thus, after interaction at 37°C, R1 contains both noninteracting iDCs and interacting iDCs. (Right) R1 of the former figure after a 2-h interaction between iDCs and DIL-stained apoptotic Jurkat cells, at 37°C. Most of the interacting iDCs (bold line) acquired DIL in this experiment as compared with unstained iDCs (dotted line). iDCs that did not bound or phagocytosed apoptotic cells are found in the area of unstained iDCs. During 2 h of interaction no passive staining of iDCs occurred as demonstrated by 2 chambers incubation (not shown). The percentage of interacting iDCs that acquired DIL, as well as, mean and median fluorescence, were measured. The interaction was performed in different iDC:Jurkat ratios and in the presence or absence of fresh serum or heat inactivated serum. In inhibition studies, iDCs were preincubated with monoclonal antibodies for 30 min before interaction. To distinguish between bound apoptotic Jurkat cells and internalized cells, we stained with anti-CD3-FITC and determine what percentage of iDCs that acquired DIL, stained for CD3. CD3, expressed on apoptotic Jurkat cells (and not on iDCs), stained only noninternalized, adhered, apoptotic Jurkat cells. Similar results were obtained upon validation of this system, using Jurkat cells stained with PKH, or double staining of CD1a-FITC, DIL-apoptotic Jurkat cells.
Mentions: We generated iDCs that were >90% CD14−CD1a+, as well as low DR, CD83, and CD86 (see below). To determine whether complement activation increased uptake of apoptotic cells, we measured uptake by iDCs by FACScan™ using similar methodology to that described (4, 7, 17), and as explained in Fig. 2 . As shown in Fig. 3 A, at a ratio of 1:1, iDC:apoptotic cell, 44.25 ± 2.21% of iDCs bound/engulfed apoptotic Jurkat cells in the absence of serum compared with 83.25 ± 4.3%, in the presence of serum (P < 0.001, triplicates, representative of six experiments). At a ratio of 1:2 the gap decreased but there was still a significant difference in all experiments, 59.2 ± 4.3% versus 93 ± 5.3%, respectively (P < 0.001). However, at 1:4 there was a further decrease and the difference was nonsignificant in one out of six experiments. At ratios of 1:8 and 1:16 there was no difference in the number of iDCs that bound/engulfed apoptotic Jurkat cells in the absence or presence of autologous fresh serum. To further evaluate the relative number of apoptotic cells bound or engulfed by iDCs, we used the median fluorescence of DIL detected in the area of the gated iDCs. As shown in Fig. 3 B, median fluorescence, at a ratio of 1:1, was increased up to eightfold in the presence of autologous serum. This difference decreased gradually upon increasing the ratio but was still elevated (1.3–1.8-fold) at a ratio of 1:16. To further verify that this effect was due to complement activation we simultaneously examined interactions with autologous heat-inactivated serum. Heat-inactivation abolished the effect of serum, the result being similar to that seen in the absence of serum (Fig. 3, C and D). The experiments were repeated using autologous plasma instead of serum, with similar results (not shown). To verify that all interacting cells were phagocytosed, staining with anti-CD3 was performed. 2 h after interaction, between 18–35% of the interacting cells still had bound Jurkat cells (at ratio 1:4). However, by 18 h after the interaction, <10% of iDCs that acquired DIL, stained for CD3.

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