Calreticulin Is at the Surface of Circulating Neutrophils and Uses CD59 as an Adaptor Molecule

Calreticulin, which has been proposed to be a C1q receptor on neutrophils, has neither a transmembrane domain nor a GPI-anchor attachment site and must utilize an adaptor molecule to attach to the plasma membrane. The expression of ecto-calreticulin on purified human neutrophils did not result from contamination by soluble or intracellular calreticulin released during cell fractionation because it was expressed on circulating neutrophils, and the expression did not increase significantly with neutrophil isolation. All neutrophils expressed calreticulin with a unimodal distribution. Treatment of neutrophils with either a cholesterol-binding agent or phosphatidylinositol-specific phospholipase C dramatically decreased ecto-calreticulin expression indicating that the adaptor molecule(s) are located in lipid rafts and have a GPI-anchor. Analysis for the co-expression of specific GPI-anchored proteins and ecto-calreticulin in cells that were deficient in specific GPI-anchored proteins, indicated that ecto-calreticulin was best associated with CD59. Calreticulin reciprocally immunoprecipited with CD59, which provided direct evidence that CD59 is an adaptor for ecto-calreticulin. Immunofluorescence and confocal microscopy demonstrated that ecto-calreticulin co-localized with a fraction of CD59 at the cell surface. Cross-linking ecto-calreticulin with antibodies induced a Ca2+ flux, which suggests that ecto-calreticulin is capable of signaling following ligand binding. Ecto-calreticulin has been associated with diverse cellular functions. An appreciation that the adaptors for ecto-calreticulin are GPI-anchored will provide a framework for understanding any common features underlying ecto-calreticulin ligation. Calreticulin, which has been proposed to be a C1q receptor on neutrophils, has neither a transmembrane domain nor a GPI-anchor attachment site and must utilize an adaptor molecule to attach to the plasma membrane. The expression of ecto-calreticulin on purified human neutrophils did not result from contamination by soluble or intracellular calreticulin released during cell fractionation because it was expressed on circulating neutrophils, and the expression did not increase significantly with neutrophil isolation. All neutrophils expressed calreticulin with a unimodal distribution. Treatment of neutrophils with either a cholesterol-binding agent or phosphatidylinositol-specific phospholipase C dramatically decreased ecto-calreticulin expression indicating that the adaptor molecule(s) are located in lipid rafts and have a GPI-anchor. Analysis for the co-expression of specific GPI-anchored proteins and ecto-calreticulin in cells that were deficient in specific GPI-anchored proteins, indicated that ecto-calreticulin was best associated with CD59. Calreticulin reciprocally immunoprecipited with CD59, which provided direct evidence that CD59 is an adaptor for ecto-calreticulin. Immunofluorescence and confocal microscopy demonstrated that ecto-calreticulin co-localized with a fraction of CD59 at the cell surface. Cross-linking ecto-calreticulin with antibodies induced a Ca2+ flux, which suggests that ecto-calreticulin is capable of signaling following ligand binding. Ecto-calreticulin has been associated with diverse cellular functions. An appreciation that the adaptors for ecto-calreticulin are GPI-anchored will provide a framework for understanding any common features underlying ecto-calreticulin ligation. Calreticulin (CRT) 1The abbreviations used are: CRT, calreticulin; eCRT, ecto-calreticulin; GPI, glycosylphosphatidyinositol; MBCD, methyl-β-cyclodextrin; PIPLC, phosphatidylinositol-specific phospholipase C; PMN, polymorphonuclear leukocytes; fMLP, formylmethionylleucylphenylalanine; BSA, bovine serum albumin; Ab, antibody; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; PNH, paroxysmal nocturnal hemoglobinuria; HBSS, Hank's balanced salt solution with calcium and magnesium; HBSS2-, HBSS without calcium and magnesium; DAF, decay accelerating factor or CD55. is best known as a calcium-binding, endoplasmic reticulum-resident, 60-kDa chaperone. The presence of CRT in the endoplasmic reticulum is due to the KDEL receptor, which interacts with the KDEL-containing C-terminal domain of CRT. In addition to its endoplasmic reticulum location, CRT has been found at other sites including its functional presence at the surface of a variety of cells. Most of these studies utilized cultured cells, such as mouse melanoma cells (1White T.K. Zhu Q. Tanzer M.L. J. Biol. Chem. 1995; 270: 15926-15929Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), human fetal fibroblasts (2Gray A.J. Park P.W. Broekelmann T.J. Laurent G.J. Reeves J.T. Stenmark K.R. Mecham R.P. J. Biol. Chem. 1995; 270: 26602-26606Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), HeLa cells (3Llewellyn D.H. Kendall J.M. Sheikh F.N. Campbell A.K. Biochem. J. 1996; 318: 555-560Crossref PubMed Scopus (51) Google Scholar), in vitro activated T cells (4Arosa F.A. de Jesus O. Porto G. Carmo A.M. de Sousa M. J. Biol. Chem. 1999; 274: 16917-16922Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), bovine aortic endothelial cells (5Goicoechea S. Orr A.W. Pallero M.A. Eggleton P. Murphy-Ullrich J.E. J. Biol. Chem. 2000; 275: 36358-36368Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), CEM, and Jurkat cells (6Seddiki N. Nato F. Lafaye P. Amoura Z. Piette J.C. Mazie J.C. J. Immunol. 2001; 166: 6423-6429Crossref PubMed Scopus (50) Google Scholar). Because there is always a fraction of dead and dying cells within a population of cultured cells, it raises the question as to whether the ecto-calreticulin (eCRT) is derived from moribund cells and stuck to the surface of viable cells. However, eCRT has been identified on the surface of freshly isolated human polymorphonuclear neutrophils (PMN) (7Eggleton P. Lieu T.S. Zappi E.G. Sastry K. Coburn J. Zaner K.S. Sontheimer R.D. Capra J.D. Ghebrehiwet B. Tauber A.I. Clin. Immunol. Immunopath. 1994; 72: 405-409Crossref PubMed Scopus (93) Google Scholar, 8Kishore U. Sontheimer R.D. Sastry K.N. Zaner K.S. Zappi E.G. Hughes G.R. Khamashta M.A. Strong P. Reid K.B. Eggleton P. Biochem. J. 1997; 322: 543-550Crossref PubMed Scopus (69) Google Scholar), a finding that we have confirmed (9Ghiran I. Tyagi S. Klickstein L.B. Nicholson-Weller A. Immunobiology. 2002; 205: 407-420Crossref PubMed Scopus (31) Google Scholar). Engaging eCRT induces different effects in different cells, e.g. for macrophages, ligation of eCRT with either complement C1q-, mannan-binding lectin-, surfactant protein A-, or surfactant protein d-opsonized apoptotic cells, induces phagocytosis of the apoptotic cell by a macrophage CD91-dependent mechanism (10Ogden C.A. de Cathelineau A. Hoffmann P.R. Bratton D. Ghebrehiwet B. Fadok V.A. Henson P.M. J. Exp. Med. 2001; 194: 781-796Crossref PubMed Scopus (949) Google Scholar, 11Vandivier R.W. Ogden C.A. Fadok V.A. Hoffmann P.R. Brown K.K. Botto M. Walport M.J. Fisher J.H. Henson P.M. Greene K.E. J. Immunol. 2002; 169: 3978-3986Crossref PubMed Scopus (439) Google Scholar). For fibroblasts, engaging eCRT by Bβ chain of fibrinogen induces mitosis (2Gray A.J. Park P.W. Broekelmann T.J. Laurent G.J. Reeves J.T. Stenmark K.R. Mecham R.P. J. Biol. Chem. 1995; 270: 26602-26606Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), whereas engaging eCRT by the collagen domain of C1q induces a pro-apoptotic, anti-mitotic effect (12Bordin S. Whitfield D. J. Immunol. 2003; 170: 667-671Crossref PubMed Scopus (19) Google Scholar). CRT, which lacks a transmembrane domain, requires an adaptor molecule that is a resident of the plasma membrane to be expressed at the cell surface. The effect of eCRT engagement would then depend to a great extent on the identity of the adaptor molecule(s) of the plasma membrane. In this study, we report that CRT is present at the plasma membrane of circulating PMN. Normal 293 cells expressed eCRT, whereas GPI-anchor-deficient 293 cells were also eCRT deficient, consistent with the putative adaptor molecule(s) for eCRT being GPI-anchored. Using immunoprecipitation and confocal microscopy, we have identified CD59 as a major adaptor protein for eCRT in PMN. Finally, cross-linking eCRT with primary and secondary antibodies induced a calcium flux in PMN, demonstrating that eCRT ligation with a natural ligand has the potential to initiate intracellular signaling. Reagents—Reagents were purchased as noted: EDTA, fMLP, BSA, protein A, protease inhibitor mixture, sodium vanadate, Tris, propidium iodide acridine orange, C5a, and fMLP (Sigma); Dextran-70 (McGaw, Irvine, CA); Ficoll-Hypaque (Amersham Biosciences); and HBSS with Ca2+ and Mg2+ and without (HBSS2-) (Invitrogen). Tris-buffered saline-Tween buffer consisted of 25 mm Tris-HCl (pH 7.6), 150 mm NaCl, 5 mm EDTA, Tween 0.1%. Antibodies—The following antibodies were used: rabbit anti-CRT peptide residues 405–417 (Stressgen, Victoria BC, Canada); chicken anti-CRT peptide residues 339–414 (Affinity Bioreagents, Golden, CO), mouse anti-CRT mAb SPA-601 (Stressgen); rabbit anti-DAF (13Nicholson-Weller A. March J.P. Rosen C.E. Spicer D.B. Austen K.F. Blood. 1985; 65: 1237-1244Crossref PubMed Google Scholar); mouse anti-CD16 mAb 3G8 (14Fleit H.B. Wright S.D. Unkeless J.C. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3275-3279Crossref PubMed Scopus (437) Google Scholar); anti-CD59 mAbs YTH 53.1 (15Davies A. Simmons D.L. Hale G. Harrison R.A. Tighe H. Lachmann P.J. Waldmann H. J. Exp. Med. 1989; 170: 637-654Crossref PubMed Scopus (535) Google Scholar), Bric229, p282, MEM 43/5 (16Stefanova I. Hilgert I. Kristofova H. Brown R. Low M.G. Horejsi V. Mol. Immunol. 1989; 26: 153-161Crossref PubMed Scopus (109) Google Scholar); anti-CD87 mAb Vim 5 (BD Biosciences); activation epitope reporter mAb CBR M1/5 for CD11b/CD18 (17Diamond M.S. Springer T.A. J. Cell Biol. 1993; 120: 545-556Crossref PubMed Scopus (456) Google Scholar); horseradish peroxidase-conjugated goat anti-mouse and -rabbit IgG (Zymed Laboratories Inc., San Francisco, CA); and fluorescently labeled secondary antibodies of “ML grade” (multiple labeling, specifically designed for simultaneous detection of two or more antibodies) and normal goat and human serum for blocking (Jackson Immuno Research, West Grove, PA). Cells—Leukocytes were derived from finger prick blood (150 μl) that was mixed with 1 ml cold HBSS2-, 2 mm EDTA, the cells pelleted by centrifugation, and the erythrocytes lysed by ammonium chloride (8.1 g/l ammonium chloride, 1.0 g/l potassium bicarbonate, and 0.037 g/l EDTA) for 5 min. The leukocytes were then washed twice in HBSS. Dextran-sedimented leukocytes were isolated from 40 ml of ACD-anti-coagulated blood obtained by venopuncture, as described (18Ghiran I. Barbashov S.F. Klickstein L.B. Tas S.W. Jensenius J.C. Nicholson-Weller A. J. Exp. Med. 2000; 192: 1797-1808Crossref PubMed Scopus (216) Google Scholar). PMN were further fractionated from the dextran-leukocyte preparation by centrifugation through Ficoll-Paque at 3000 × g for min. 293 fetal cells were with an T and to a normal GPI-anchor cell and a GPI-anchor-deficient cell were by L.B. Springer T.A. S.F. J.M. J. S. R. Springer V. Scholar). cells, were provided by S. of which were and were for with as in in buffer at by two and for with secondary at a by the were washed and in a (BD In the at were and the were Immunofluorescence and the were in at for in HBSS primary and secondary antibodies cell were by with HBSS for min. The of primary antibodies was and the of secondary were as by the with secondary PMN were washed twice and in HBSS For of eCRT and CD59 in cells, PMN were to to the for with for washed and in were with a on an with and For a 40 × was were further For confocal cells were the in and on at were at the of × a confocal on a with a × were further The of the was that there was from the with were and PMN were washed twice in HBSS and lysed in buffer mm Tris-HCl (pH 7.6), 150 mm NaCl, 5 mm EDTA, and protease inhibitor for on The was for at × and the was for with protein for with mAb by The was then for with protein with either anti-CRT mAb or anti-CD59 or anti-CD16 were washed in buffer and in buffer for 5 min. were on on and with in buffer with Tween for 1 at were with anti-CRT mAb anti-CD59 or anti-CD16 mAb for at and with horseradish peroxidase-conjugated secondary for an min. were washed in buffer with Tween and with West and to purified PMN were in at a of and with the for in at by two in cold were then in HBSS and with either anti-CD59 mAb or anti-CRT and antibodies for at the cells were washed with cold buffer then to for to The addition of secondary was Ca2+ by at in a San with at The were with Calreticulin on the and a the of PMN from to as by propidium cells proteins found in the which to the surface of cells. the presence of an endoplasmic protein at the cell surface. this we purified PMN and for CRT expression during the The expression of eCRT during the of the cells eCRT was expressed on PMN derived from finger prick blood CRT lacks a transmembrane domain and has that would lipid to membrane attachment B. P. F. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). to eCRT requires an adaptor molecule for expression at the plasma membrane. the eCRT and its adaptor molecule were of the adaptor expression be by in eCRT eCRT by the or of eCRT expression provide as to adaptor protein for eCRT, we PMN to fMLP from to × and the eCRT expression was by The of fMLP in of eCRT and also to significantly eCRT expression as by in of and CRT calcium and it has a whether eCRT were to an adaptor in a we the effect of PMN to for at did not significantly eCRT expression result also of CRT to the cell consistent with the from did a in HBSS at as U. Sontheimer R.D. Sastry K.N. Zaner K.S. Zappi E.G. Hughes G.R. Khamashta M.A. Strong P. Reid K.B. Eggleton P. Biochem. J. 1997; 322: 543-550Crossref PubMed Scopus (69) Google Scholar, P. Ghebrehiwet B. Coburn J.P. Sastry K.N. Zaner K.S. Tauber A.I. Blood. 1994; PubMed Google Scholar), and the was by the of PMN to for that the on and not and C the of a for the adaptor molecule(s) for eCRT to the cell we GPI-anchored proteins were PMN with or buffer and the expression of is a of with a which its and a which other cholesterol-binding not with the plasma from the surface of the cell membrane by specifically the cell of in a lipid proteins from lipid rafts S. Biochem. J. PubMed Scopus Google Scholar). with the decreased the expression of eCRT on PMN by more a and that the adaptor for eCRT in lipid rafts are in GPI-anchored proteins and a transmembrane proteins in Biochem. PubMed Scopus Google Scholar). is an that specifically GPI-anchored proteins at lipid the transmembrane and GPI-anchored proteins in lipid PMN were with for and then with chicken anti-CRT or mAb is known to be a GPI-anchored protein on PMN C.E. R. M. D. PubMed Scopus Google Scholar). of PMN decreased expression of eCRT by is not in the GPI-anchored proteins from cells C.E. R. M. D. PubMed Scopus Google and as by the of in this GPI-anchored of The of eCRT by the of a GPI-anchored adaptor for CRT. this we utilized a of 293 cells that been and antibodies GPI-anchored proteins L.B. Springer T.A. S.F. J.M. J. S. R. Springer V. Scholar). CD59, a known GPI-anchored a normal expression on the cells, and eCRT was also expressed a and The GPI-anchor-deficient cells to CD59 and to eCRT and provided further evidence that eCRT expression was to the expression of GPI-anchored proteins, it did not the GPI-anchored because GPI-anchored proteins were from the cells. eCRT the of CD59 and on the PMN from GPI-anchored proteins on including CD59, and and eCRT from with are deficient in the two complement proteins and CD59 and be deficient in other GPI-anchored proteins as eCRT expression in the PMN from a normal and two The PMN were not and eCRT was expressed a and were consistent did not the finding that either or CD59 be the adaptor for we a more PMN were not and In this the PMN were consistent with CD59 being the adaptor and as a major adaptor and were confirmed the cell which were and with of and of CRT and CD59 of CRT with CD59 expression on the PMN from PMN and cells to there were any eCRT and CD59. eCRT rabbit anti-CRT and the either anti-CD59 mAb YTH 53.1 or anti-CD16 CD59 with whereas did not the immunoprecipitation with anti-CD59 mAb, we eCRT by in a of the as intracellular CRT not anti-CD59 mAb was in The other anti-CD59 mAbs that to eCRT, including YTH 53.1 and which epitope and p282, which epitope L.B. Springer S.F. J.M. J. S. R. Springer V. Scholar). that 1 and of CD59, not epitope eCRT with a of CD59 at the of the microscopy it is to the presence of two or more proteins in the at the in a used this to the of eCRT and CD59 on human PMN. purified cells were in HBSS with either with rabbit anti-CRT or with mAb anti-CD59 for on by two and by and secondary The that eCRT is present at the surface of PMN and with a fraction of CD59 were obtained with other anti-CD59 a we used mAb CBR which an epitope of CD11b/CD18 (17Diamond M.S. Springer T.A. J. Cell Biol. 1993; 120: 545-556Crossref PubMed Scopus (456) Google In PMN, of the proteins not to at the of the cell the of as is demonstrated in In to any cells were and confocal microscopy by PMN is (eCRT) and the surface and two through the of the which of and that the cell was not at the of and Most of the from the at the cell surface whereas were either or and All the cells more CD59 on surface eCRT, different different of eCRT of PMN in PMN were as and in with for 5 min. were then washed and in on were a confocal and used to the PMN PMN a surface for CD59 and eCRT with intracellular and was in In and in CD59 and eCRT were in whereas in and in were in at the plasma Cross-linking of eCRT on PMN a to that by cross-linking of GPI-anchored proteins intracellular signaling. The signaling used CD59 is a Ca2+ flux and the activation of Horejsi V. J. Immunol. 1993; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cross-linking eCRT would a cells were with for washed in cold and mixed with either anti-CD59 mAb or anti-CRT for at the cells were washed with cold buffer then to for to addition of secondary a cells were with by secondary Ab, and flux was Cross-linking eCRT induced a Ca2+ flux cross-linking CD59 and is that there is for cross-linking there is CD59, for as in the confocal In for the adaptor molecule(s) for eCRT to the plasma we found evidence that the fMLP and significantly the expression of eCRT on PMN, CRT being in a intracellular with and the of GPI-anchored adaptor molecule(s) for eCRT that the cells, which also eCRT was further evidence that for eCRT were GPI-anchored However, and the evidence provided by the from was is to be an with the GPI-anchor of and CD59, and the of other proteins as J.M. J. and the San Scholar), other proteins are we that there was an with CD59 and eCRT CD59 and eCRT was by immunoprecipitation to epitope 2 of CD59, not to 1 and eCRT L.B. Springer S.F. J.M. J. S. R. Springer V. Scholar). All these mAb PMN not it is that eCRT was by mAb to 1 and and these mAbs the The confocal microscopy that not of the eCRT with CD59 not the of the other adaptor studies of the 293 cells and that must be and we specifically from studies with that is not not CD59 was described as a complement protein that interacts with and to cell by the complement (15Davies A. Simmons D.L. Hale G. Harrison R.A. Tighe H. Lachmann P.J. Waldmann H. J. Exp. Med. 1989; 170: 637-654Crossref PubMed Scopus (535) Google Scholar, M. Biochem. PubMed Scopus Google Scholar, J. Clin. 1989; PubMed Scopus Google Scholar, S. M. J. A. Lachmann P.J. J. Exp. Med. PubMed Scopus Google Scholar, P.J. J. Immunol. Google Scholar). is whether eCRT to CD59 or the complement of the depend on CD59 to from M. A. H. H. N. N. H. K. J. Med. PubMed Scopus Google Scholar), and the that of eCRT (9Ghiran I. Tyagi S. Klickstein L.B. Nicholson-Weller A. Immunobiology. 2002; 205: 407-420Crossref PubMed Scopus (31) Google suggests that eCRT is not for CD59 complement the other the the of the complement on the cell then eCRT signaling to the cell J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Nicholson-Weller A. J. Clin. 1993; PubMed Scopus Google Scholar, F. H. S. M.L. J. Immunol. 1993; Google Scholar). The calcium flux that we and M.S. Park K. J. Mol. Biol. 2000; PubMed Scopus Google have eCRT is is consistent with the finding that cross-linking any of the GPI-anchored proteins on PMN and induces a calcium flux Horejsi V. J. Immunol. 1993; PubMed Scopus Google Scholar). has been associated with eCRT in human macrophages, as a receptor for soluble CRT S. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, B. 2001; PubMed Scopus Google Scholar), and as of a with eCRT that for the of and by collagen (10Ogden C.A. de Cathelineau A. Hoffmann P.R. Bratton D. Ghebrehiwet B. Fadok V.A. Henson P.M. J. Exp. Med. 2001; 194: 781-796Crossref PubMed Scopus (949) Google Scholar, 11Vandivier R.W. Ogden C.A. Fadok V.A. Hoffmann P.R. Brown K.K. Botto M. Walport M.J. Fisher J.H. Henson P.M. Greene K.E. J. Immunol. 2002; 169: 3978-3986Crossref PubMed Scopus (439) Google Scholar). However, direct of eCRT to has been demonstrated to PMN not and would a different molecule as adaptor S. K. K. E.G. J. J.C. P. S. G. J. A. J. C.E. Springer T.A. M.J. P. S.F. J.M. J. S. R. Springer Cell Scholar). 293 cells CD59 and of the of which is not GPI-anchored S. K. K. E.G. J. J.C. P. S. G. J. A. J. C.E. Springer T.A. M.J. P. S.F. J.M. J. S. R. Springer Cell Scholar). In the GPI-anchor-deficient cells we found that eCRT was also deficient in PMN and in the fetal cell not as an adaptor for of human also decreased expression of cell surface CRT not and this were also for macrophages, it that must in a with eCRT and a GPI-anchored have found that receptor is a functional receptor on PMN and receptor on erythrocytes for the collagen domain of complement C1q and the mannan-binding (18Ghiran I. Barbashov S.F. Klickstein L.B. Tas S.W. Jensenius J.C. Nicholson-Weller A. J. Exp. Med. 2000; 192: 1797-1808Crossref PubMed Scopus (216) Google Scholar, L.B. Barbashov S. Nicholson-Weller A. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S.W. Klickstein L.B. Nicholson-Weller A. J. Immunol. 1999; Google Scholar). However, the of isolated or CRT to C1q is also to the B. J. Immunol. PubMed Scopus Google Scholar, A. Reid K.B. Biochem. J. Google Scholar, R. S. Reid J. Exp. Med. PubMed Scopus Google Scholar, R. Jensenius J. Immunol. 1993; Google Scholar). In C1q collagen to purified CRT requires as to L.B. Barbashov S. Nicholson-Weller A. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Biochem. J. 1993; PubMed Scopus Google Scholar); whereas C1q to sites in CRT in normal buffer U. Sontheimer R.D. Sastry K.N. Zaner K.S. Zappi E.G. Hughes G.R. Khamashta M.A. Strong P. Reid K.B. Eggleton P. Biochem. J. 1997; 322: 543-550Crossref PubMed Scopus (69) Google Scholar). C1q induces PMN to and A.J. J. Immunol. Google Scholar, Nicholson-Weller A. Barbashov S.F. Tas S.W. Klickstein L.B. and 2000; PubMed Scopus Google Scholar). The PMN receptor that this is not Nicholson-Weller A. Barbashov S.F. Tas S.W. Klickstein L.B. and 2000; PubMed Scopus Google Scholar). have found that rabbit by PMN P. Ghebrehiwet B. Coburn J.P. Sastry K.N. Zaner K.S. Tauber A.I. Blood. 1994; PubMed Google Scholar), a finding we have confirmed anti-CRT a peptide of residues not that eCRT be the receptor for this PMN In studies eCRT and intracellular CRT the on However, rabbit the protein isolated from plasma B. J. Immunol. PubMed Scopus Google with a of whereas calreticulin has a of (7Eggleton P. Lieu T.S. Zappi E.G. Sastry K. Coburn J. Zaner K.S. Sontheimer R.D. Capra J.D. Ghebrehiwet B. Tauber A.I. Clin. Immunol. Immunopath. 1994; 72: 405-409Crossref PubMed Scopus (93) Google Scholar). it that C1q cellular CRT, the and eCRT there is a for CRT in its expression is to and any putative intracellular and eCRT in PMN S. M. M. 2002; PubMed Scopus Google Scholar). Finally, the functional of ligand to eCRT will depend the adaptor proteins of GPI-anchored proteins as the primary adaptors for eCRT will provide for studies in this

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