Identification of Two Distinct Mechanisms of Phagocytosis Controlled by Different Rho GTPases
10.1126/science.282.5394.1717
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

The complement and immunoglobulin receptors are the major phagocytic receptors involved during infection. However, only immunoglobulin-dependent uptake results in a respiratory burst and an inflammatory response in macrophages. Rho guanosine triphosphatases (molecular switches that control the organization of the actin cytoskeleton) were found to be essential for both types of phagocytosis. Two distinct mechanisms of phagocytosis were identified: Type I, used by the immunoglobulin receptor, is mediated by Cdc42 and Rac, and type II, used by the complement receptor, is mediated by Rho. These results suggest a molecular basis for the different biological consequences that are associated with phagocytosis.

Bibliography

Caron, E., & Hall, A. (1998). Identification of Two Distinct Mechanisms of Phagocytosis Controlled by Different Rho GTPases. Science, 282(5394), 1717–1721.

Authors 2
  1. Emmanuelle Caron (first)
  2. Alan Hall (additional)
References 44 Referenced 763
  1. Allen L.-A. H., Aderem A., Curr. Opin. Immunol. 8, 36 (1996). (10.1016/S0952-7915(96)80102-6) / Curr. Opin. Immunol. by Allen L.-A. H. (1996)
  2. Newman S. L., Mikus L. K., Tucci M. A., J. Immunol. 146, 967 (1991); (10.4049/jimmunol.146.3.967) / J. Immunol. by Newman S. L. (1991)
  3. Allen L.-A. H., Aderem A., J. Exp. Med. 184, 627 (1996). (10.1084/jem.184.2.627) / J. Exp. Med. by Allen L.-A. H. (1996)
  4. Kaplan G., Scand. J. Immunol. 6, 797 (1977). (10.1111/j.1365-3083.1977.tb02153.x) / Scand. J. Immunol. by Kaplan G. (1977)
  5. Aderem A. A., Wright S. D., Silverstein S. C., Cohn Z. A., J. Exp. Med. 161, 617 (1985). (10.1084/jem.161.3.617) / J. Exp. Med. by Aderem A. A. (1985)
  6. Wright S. D., Silverstein S. C., ibid. 158, 2016 (1983); / ibid. by Wright S. D. (1983)
  7. ; K. Yamamoto and R. B. Johnston ibid. 159 405 (1984). (10.1084/jem.159.2.405)
  8. Scholl P. R., Ahern D., Geha R. S., J. Immunol. 149, 1751 (1992); (10.4049/jimmunol.149.5.1751) / J. Immunol. by Scholl P. R. (1992)
  9. Stein M., Gordon S., Eur. J. Immunol. 21, 431 (1991). (10.1002/eji.1830210227) / Eur. J. Immunol. by Stein M. (1991)
  10. 10.1126/science.279.5350.509
  11. 10.1016/0092-8674(92)90163-7
  12. ; A. J. Ridley H. F. Paterson C. L. Johnston D. Diekmann A. Hall ibid. p. 401;
  13. Kozma R., Ahmed S., Best A., Lim L., Mol. Cell. Biol. 15, 1942 (1995). (10.1128/MCB.15.4.1942) / Mol. Cell. Biol. by Kozma R. (1995)
  14. 10.1016/0092-8674(95)90370-4
  15. Perona R., et al., Genes Dev. 11, 463 (1997). (10.1101/gad.11.4.463) / Genes Dev. by Perona R. (1997)
  16. Coso O. A., et al., Cell 81, 1137 (1995); (10.1016/S0092-8674(05)80018-2) / Cell by Coso O. A. (1995)
  17. ; A. Minden A. Lin F.-X. Claret A. Abo M. Karin ibid. p. 1147; H. Teramoto et al. J. Biol. Chem. 271 25731 (1996);
  18. 10.1038/353668a0
  19. Gabig T. G., Crean C. D., Mantel P. L., Rosli R., Blood 85, 804 (1995). (10.1182/blood.V85.3.804.bloodjournal853804) / Blood by Gabig T. G. (1995)
  20. cDNAs encoding human FcγRIIA CD11b and CD18 were subcloned into pRK5. Subconfluent serum-starved Swiss 3T3 cells were prepared as described [
  21. Lamarche N., et al., Cell 87, 519 (1996); (10.1016/S0092-8674(00)81371-9) / Cell by Lamarche N. (1996)
  22. ]. Eukaryotic expression vectors (0.1 mg/ml) encoding phagocytic receptors and Myc-tagged GTPase constructs were microinjected into the nucleus of at least 50 cells over a period of 10 min in a temperature- and CO 2 -controlled chamber. Cells were returned to the incubator for 3 hours for optimal expression incubated for 20 min at 4°C with monoclonal antibodies (mAbs) (10 μg/ml) to human CD11b (clone 44 Pharmingen San Diego CA) or to human FcγRIIA (clone IV.3 Medarex Annandale NJ) washed in ice-cold serum-free medium and further incubated at 37°C with donkey anti-mouse IgG (15 μg/ml). Cells were rinsed in serum-free medium fixed for 10 min in freshly prepared cold 4% (w/v) paraformaldehyde and processed for immunofluorescence as described (9). Biotin-conjugated mAb 9E.10 followed by cascade blue–conjugated avidin was used to detect Myc-tagged constructs. Anti-CD11b or anti-FcγRIIA followed by fluorescein isothiocyanate (FITC)–conjugated F(ab′) 2 antimouse IgG was used to detect receptor expression. Rhodamine-labeled phalloidin (Sigma) was used to visualize F-actin. To block the nonspecific binding of antibodies to the Fc receptor we performed incubations with antibodies when appropriate in the presence of excess human IgGs.
  23. COS-1 cells were transfected by the DEAE-dextran method as described [
  24. 10.1126/science.7652575
  25. ]. Plasmid amounts in each 10-cm petri dish were as follows: 4 μg of pRK5-FcγRIIA or 2 μg of pRK5-CD11b and 2 μg of pRK5-CD18 with either 1 μg of pRK5myc 1 μg of pEFmyc-C3 transferase or one of the Rho Rac and Cdc42 constructs (1 μg). Twenty-four hours later cells were serum-starved for 16 hours before phagocytic challenge and immunofluorescence. Sheep RBCs (Cappel Thame UK) were coated with IgG (IgG-RBCs) or with C3bi (C3bi-RBCs) as follows. RBCs in gelatin veronal buffer were incubated in the presence of subagglutining concentrations of IgG or IgM antibodies to rabbit RBCs (Cappel). RBCs were then washed and IgM-coated RBCs were further opsonized by incubation with 10% (v/v) C5-deficient human serum for 20 min at 37°C. Under these conditions C3b is rapidly fixed to IgM-coated RBCs and is completely converted into C3bi [
  26. Newman S. L., Mikus L. K., J. Exp. Med. 161, 1414 (1985); (10.1084/jem.161.6.1414) / J. Exp. Med. by Newman S. L. (1985)
  27. ]. Opsonization was checked by fluorescence after incubation with FITC-conjugated anti-rabbit IgG or with goat anti-C3 (Sigma) followed by FITC-conjugated anti-goat IgG. All IgG-RBCs and C3bi-RBCs appeared to be uniformly labeled. After a final wash the RBCs were suspended in Hepes-buffered Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL) and added to cells at a ratio of ∼10 RBCs per cell. For phagocytosis assays complement-opsonized RBCs were allowed to interact for 20 min at 37°C with transfected COS cells. IgG-opsonized RBCs were allowed to adhere to cells for 15 min at 4°C then unbound RBCs were washed off and phagocytes were further incubated for 15 min at 37°C in Hepes-buffered DMEM. After fixation and immunofluorescence transfected COS cells were scored for their ability to bind or phagocytose RBCs (14). COS cells were examined for phagocytic receptor (control) or Myc expression (cotransfection with Myc-tagged constructs) and only the positive cells were analyzed for RBC binding or phagocytosis. Positive cells represented ∼20% of the total number of COS cells per cover slip that is ≥100 cells per cover slip. Rhodamine-conjugated donkey anti-rabbit IgGs were used to detect RBCs. An attachment was defined by the ability of injected or transfected cells to bind one or more RBCs. Phagocytosis was defined by the ability of transfected cells to internalize one or more RBCs. No difference was observed in the total number of particles binding to the different transfectant populations.
  28. S. Greenberg J. el Khoury
  29. Kaplan E., Silverstein S. C., J. Immunol. Methods 139, 115 (1991); (10.1016/0022-1759(91)90358-M) / J. Immunol. Methods by Kaplan E. (1991)
  30. . Surface-bound particles appear to be small have a crenated appearance are readily distinguished from internalized particles that are enclosed in a vacuole and have a swollen circular appearance. In our hands this method and the counting of RBC stained pre- and postpermeabilization with different conjugates resulted in similar quantitative results in COS-1 and J774 cells.
  31. 10.1038/375500a0
  32. . Pretreatment (2 hours) of J774.A1 cells with toxin B inhibited (in a dose-dependent manner) both CR3- and FcγR-mediated phagocytosis [maximal reductions of 92 and 88% respectively with toxin B (10 ng/ml)] but had no effect on the initial binding of opsonized targets to J774 macrophages.
  33. J774.A1 cells grown in DMEM and supplemented with 10% heat-inactivated fetal calf serum (Sigma) and 5% penicillin/streptomycin (Gibco-BRL) were plated overnight on acid-washed glass cover slips (13 mm in diameter) in four-well plates at a density of 10 5 cells per milliliter in each well. Cells were serum-starved immediately before microinjection. Biotin dextran (2.5 mg/ml) (Molecular Probes Eugene OR) was injected alone or with eukaryotic expression vectors (0.1 mg/ml) encoding Myc-tagged GTPase constructs into the nucleus of at least 50 cells over a period of 10 min. Cells were returned to the incubator for 3 hours for optimal expression. RBCs were opsonized and phagocytic assay and immunofluorescence were performed as described (13). The two modifications that we introduced were (i) the preactivation of J774 cells for 15 min at 37°C with phorbol 12-myristate 13-acetate (150 ng/ml) in serum-free medium before the phagocytic challenge with CR3 targets and (ii) the visualization of microinjected cells after staining with cascade blue–conjugated avidin (Molecular Probes). To block nonspecific binding of antibodies to the Fc receptor we performed incubations with antibodies in the presence of excess human or murine IgGs. All injected (cascade blue positive) J774.A1 control cells or all Myc-expressing macrophages were assessed (that is ≥50 cells per condition). Microinjection did not affect viability or morphology nor did it interfere with the cell's ability to bind targets. The percentage of phagocytosis-competent cells was similar in uninjected cells and cells that were injected with biotin dextran.
  34. Nagata K., Driessens M., Lamarche N., Gorski J., Hall A., J. Biol. Chem. 273, 15453 (1998). (10.1074/jbc.273.25.15453) / J. Biol. Chem. by Nagata K. (1998)
  35. 10.1084/jem.186.9.1487
  36. D. J. Hackam O. D. Rotstein A. Schreiber W.-J. Zhang S. Grinstein ibid. p. 955.
  37. L. Van Aelst and C. D'Souza-Schorey Genes Dev. 11 2295 (1997). (10.1101/gad.11.18.2295)
  38. Rhoades K. L., Golub S. H., Economou J. S., J. Biol. Chem. 267, 22102 (1992); (10.1016/S0021-9258(18)41641-9) / J. Biol. Chem. by Rhoades K. L. (1992)
  39. 10.1038/372739a0
  40. Fadok V. A., et al., J. Clin. Invest. 101, 890 (1998); (10.1172/JCI1112) / J. Clin. Invest. by Fadok V. A. (1998)
  41. Mosser D. M., Edelson P. J., J. Immunol. 135, 2785 (1985); (10.4049/jimmunol.135.4.2785) / J. Immunol. by Mosser D. M. (1985)
  42. ; Nature 327 329 (1987); T. J. Holzer K. E. Nelson R. G. Crispen B. R. Andersen. Infect. Immun. 51 514 (1986); (10.1128/iai.51.2.514-520.1986)
  43. Schlesinger L. S., Horwitz M. A., J. Clin. Invest. 85, 1304 (1990). (10.1172/JCI114568) / J. Clin. Invest. by Schlesinger L. S. (1990)
  44. This work was generously supported by a Cancer Research Campaign (UK) program grant. E.C. was the recipient of a Training and Mobility of Researchers fellowship from the European Union. We thank B. Seed for human FcγRIIA A. Law for CD11b and CD18 B. Wren for purified toxin B and C. D. Nobes and L. M. Machesky for their constructive help and useful discussions throughout this work.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:44 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 12:23 a.m.)
Indexed 3 weeks ago (Aug. 7, 2025, 5:05 p.m.)
Issued 26 years, 9 months ago (Nov. 27, 1998)
Published 26 years, 9 months ago (Nov. 27, 1998)
Published Print 26 years, 9 months ago (Nov. 27, 1998)
Funders 0

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@article{Caron_1998, title={Identification of Two Distinct Mechanisms of Phagocytosis Controlled by Different Rho GTPases}, volume={282}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.282.5394.1717}, DOI={10.1126/science.282.5394.1717}, number={5394}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Caron, Emmanuelle and Hall, Alan}, year={1998}, month=nov, pages={1717–1721} }