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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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Interaction of apoptotic cells with iDCs is facilitated by autologus complement and complement receptors. After 2 h of incubation with apoptotic cells, iDCs were analyzed for interaction using flow cytometry as explained in Fig. 2. (A) The percentage of iDCs that interacted with DIL-stained, iC3b-opsonized, apoptotic cells, is given for different ratios of iDCs:apoptotic cells. (B) Median fluorescence of DIL as expressed in iDCs is given for different ratios. (C and D) Heat-inactivation abolished the effect of serum seen in A and B, and brought it to the level of interaction seen in the absence of serum. Average of triplicates measures is given (error bars). Representative of six experiments. (E) iDCs express CD11b/CD18 and CD11c/CD18 (iDC, bold line). Marked down-regulation of both CD11b/CD18 and CD11c/CD18 but not in CD1a, was observed after interaction with iC3b-opsonized apoptotic cells (iDC+apo, bold line). (F) 91% of iC3b-opsonized apoptotic cells were cleared by iDCs (None). No significant increase in the remaining noncleared apoptotic cells was seen when control mouse IgG was added (mIgG), compared with significant, 53% inhibition, in clearance seen when αCD11b (P < 0.05), or αCD11c (46% inhibition, P < 0.05), or both (59% inhibition, P < 0.01), were added. Representative of six experiments. DC, dendritic cells; MF, median fluorescence; NS, no serum, i.e., apoptotic cells were not exposed to fresh serum from iDC donor; S, serum, i.e., apoptotic cells were exposed to 15% fresh serum from iDC donor.
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fig3: Interaction of apoptotic cells with iDCs is facilitated by autologus complement and complement receptors. After 2 h of incubation with apoptotic cells, iDCs were analyzed for interaction using flow cytometry as explained in Fig. 2. (A) The percentage of iDCs that interacted with DIL-stained, iC3b-opsonized, apoptotic cells, is given for different ratios of iDCs:apoptotic cells. (B) Median fluorescence of DIL as expressed in iDCs is given for different ratios. (C and D) Heat-inactivation abolished the effect of serum seen in A and B, and brought it to the level of interaction seen in the absence of serum. Average of triplicates measures is given (error bars). Representative of six experiments. (E) iDCs express CD11b/CD18 and CD11c/CD18 (iDC, bold line). Marked down-regulation of both CD11b/CD18 and CD11c/CD18 but not in CD1a, was observed after interaction with iC3b-opsonized apoptotic cells (iDC+apo, bold line). (F) 91% of iC3b-opsonized apoptotic cells were cleared by iDCs (None). No significant increase in the remaining noncleared apoptotic cells was seen when control mouse IgG was added (mIgG), compared with significant, 53% inhibition, in clearance seen when αCD11b (P < 0.05), or αCD11c (46% inhibition, P < 0.05), or both (59% inhibition, P < 0.01), were added. Representative of six experiments. DC, dendritic cells; MF, median fluorescence; NS, no serum, i.e., apoptotic cells were not exposed to fresh serum from iDC donor; S, serum, i.e., apoptotic cells were exposed to 15% fresh serum from iDC donor.

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)

Interaction of apoptotic cells with iDCs is facilitated by autologus complement and complement receptors. After 2 h of incubation with apoptotic cells, iDCs were analyzed for interaction using flow cytometry as explained in Fig. 2. (A) The percentage of iDCs that interacted with DIL-stained, iC3b-opsonized, apoptotic cells, is given for different ratios of iDCs:apoptotic cells. (B) Median fluorescence of DIL as expressed in iDCs is given for different ratios. (C and D) Heat-inactivation abolished the effect of serum seen in A and B, and brought it to the level of interaction seen in the absence of serum. Average of triplicates measures is given (error bars). Representative of six experiments. (E) iDCs express CD11b/CD18 and CD11c/CD18 (iDC, bold line). Marked down-regulation of both CD11b/CD18 and CD11c/CD18 but not in CD1a, was observed after interaction with iC3b-opsonized apoptotic cells (iDC+apo, bold line). (F) 91% of iC3b-opsonized apoptotic cells were cleared by iDCs (None). No significant increase in the remaining noncleared apoptotic cells was seen when control mouse IgG was added (mIgG), compared with significant, 53% inhibition, in clearance seen when αCD11b (P < 0.05), or αCD11c (46% inhibition, P < 0.05), or both (59% inhibition, P < 0.01), were added. Representative of six experiments. DC, dendritic cells; MF, median fluorescence; NS, no serum, i.e., apoptotic cells were not exposed to fresh serum from iDC donor; S, serum, i.e., apoptotic cells were exposed to 15% fresh serum from iDC donor.
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fig3: Interaction of apoptotic cells with iDCs is facilitated by autologus complement and complement receptors. After 2 h of incubation with apoptotic cells, iDCs were analyzed for interaction using flow cytometry as explained in Fig. 2. (A) The percentage of iDCs that interacted with DIL-stained, iC3b-opsonized, apoptotic cells, is given for different ratios of iDCs:apoptotic cells. (B) Median fluorescence of DIL as expressed in iDCs is given for different ratios. (C and D) Heat-inactivation abolished the effect of serum seen in A and B, and brought it to the level of interaction seen in the absence of serum. Average of triplicates measures is given (error bars). Representative of six experiments. (E) iDCs express CD11b/CD18 and CD11c/CD18 (iDC, bold line). Marked down-regulation of both CD11b/CD18 and CD11c/CD18 but not in CD1a, was observed after interaction with iC3b-opsonized apoptotic cells (iDC+apo, bold line). (F) 91% of iC3b-opsonized apoptotic cells were cleared by iDCs (None). No significant increase in the remaining noncleared apoptotic cells was seen when control mouse IgG was added (mIgG), compared with significant, 53% inhibition, in clearance seen when αCD11b (P < 0.05), or αCD11c (46% inhibition, P < 0.05), or both (59% inhibition, P < 0.01), were added. Representative of six experiments. DC, dendritic cells; MF, median fluorescence; NS, no serum, i.e., apoptotic cells were not exposed to fresh serum from iDC donor; S, serum, i.e., apoptotic cells were exposed to 15% fresh serum from iDC donor.
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