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Expression and function of sialoadhesin in rat alveolar macrophages
Immunology Letters 71 (2000) 167 – 170
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Expression and function of sialoadhesin in rat alveolar macrophages Klemens Frei a,b,*, Christina Steger b, Puchit Samorapoompichit a, Trevor Lucas b, Othmar Fo¨rster b b
a Institute of Histology and Embryology, Uni6ersity of Vienna, Schwarzspanierstraße 17, A-1090 Vienna, Austria Institute of General and Experimental Pathology, Uni6ersity of Vienna, Schwarzspanierstraße 17, A-1090 Vienna, Austria
Received 25 October 1999; received in revised form 5 December 1999; accepted 7 December 1999
Abstract Alveolar macrophages (Amf) represent an immunologically distinct sub-population within the reticuloendothelial system. Phagocytosis and possibly antigen presentation by Amf are essential components of specific and innate primary immune defence processes against inhaled material. The mf-restricted sheep erythrocyte receptor sialoadhesin (Sn) is a member of the immunglobulin superfamily and binds specifically to sialic acid-containing structures such as selectins and was originally identified as the sheep erythrocyte receptor (SER) responsible for sialic acid-dependent binding of native sheep erythrocytes (SE) to resident murine bone marrow macrophages in rosetting assays. Sn expression has been demonstrated on murine and rat mf in lymphatic organs and is recognised by the monoclonal antibody (mAb) ED3 in the rat. In addition, sialic acid-dependent receptor (SAR) activities that mediate rosette formation of alveolar, peritoneal, splenic and bone marrow-resident rat mf with SE pretreated with gangliosides and SER-like activities between native SE and trypsinised Amf, have been described. The binding activities of both SAR and Sn show similar characteristics suggesting that these molecules are closely structurally related or identical. To clarify the relationship between Sn, SAR and SER-like activities, the binding of mAb ED3 to isolated rat Amf was investigated by flow cytometry and rosetting assays. It is demonstrated that rat Amf express Sn and evidence is provided that SAR and SER-like activities are mediated by Sn. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Sialic acid-dependent recptor (SAR); Sialoadhesin (Sn); Immunglobulin superfamily; ED3; Rosette assay
1. Introduction Sialic acid-dependent receptor (SAR) binds sialic acid-containing structures and is responsible for cellular interactions between rat mf (alveolar, peritoneal, splenic and bone marrow) and sheep erythrocytes (SE) pretreated with gangliosides (SEG) [1,2]. Treatment of SE with neuraminidase as well as addition of sialic acid-containing oligosaccharides inhibits SAR-activity [3]. A SAR called sialoadhesin (Sn) was initially characterized from murine resident bone marrow mf binding of native SE which is inhibited by pretreatment with neuraminidase or gangliosides [4]. Sn expression has * Corresponding author. Tel.: +43-1-427761346; fax: +43-142779613. E-mail address: [email protected] (K. Frei)
been demonstrated on murine mf in lymphatic organs and Kupffer cells [5]. The distribution of this receptor in lymphatic organs of the rat has also been characterised by the monoclonal antibody (mAb) ED3 [6]. Cloning of murine Sn demonstrated this receptor as a new member of the immunglobulin superfamily [7,8] with 17 immunoglobulin-like domains closely related to CD22 [9,10] and myelin-associated glycoprotein [11– 13]. Binding activities of both SAR and Sn is independent of divalent cations [2,5,14], show similar pH characteristics [2,5] and affinity for different gangliosides [2,4,15]. SAR and Sn activities are also not inhibited by metabolic inhibitors and do not trigger phagocytosis [5,12]. The expression of these receptors has been inversely correlated to major histocompatibility class II (Ia) levels [5,16] and is similarly regulated by hormones and cytokines [1,5,17]. These observations led to the
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K. Frei et al. / Immunology Letters 71 (2000) 167–170
conclusion that both receptors show a close functional relationship or identity. The aim of the present study was to investigate the expression and function of Sn on rat Amf. The expression of Sn was demonstrated by flow cytometrical analyses with the mAb ED3. Functional inhibition by this antibody in rosetting assays provides evidence that SAR and SER-like activities are mediated by Sn.
2. Materials and methods
2.1. Cell preparation Amf were isolated from 150 to 160 g male Louvain rats (Himberg, Austria). Amf were harvested by bronchoalveolar lavage in Hank’s buffered saline solution (HBSS buffer) supplemented with 0.25% EDTA and 2% fetal calf serum (Flow laboratories, Irvine, Scotland). Cell viabilities were estimated by trypan blue exclusion and purity of Amf populations was routinely assessed as \ 99% by morphology and non-specific esterase staining as described previously. For SER-like assays, Amf were resuspended in a 0.05% trypsin solution (Gibco BRL, UK) and incubated for 15 min at 37°C. After washing in HBSS, cells were resuspended in hepes-buffered RPMI 1640 (Gibco BRL, UK). Erythrocytes from male sheep (Immuno, Austria) stored in Alserver’s solution at 4°C for up to 5 days, were incubated for 1 h at 37°C in a 100 mg/ml solution of bovine brain gangliosides (Sigma, St. Louis, MO), primarily containing GM1 and GD1a, pretreated in an ultrasound device.
2.2. SAR and SER-like assays The mouse mAb ED3 recognising rat Sn was generously provided by Christine Dijkstra, Free University of Amsterdam, Holland. Murine IgG2a isotype control mAb was purchased from DAKO (Glostrup, Denmark). Purified mAb OX42 directed against rat CD11b/ c was purchased from Pharmingen (San Diego, CA). All antibodies were absorbed with SE to remove residual binding activity. A total of 50 ml suspensions of Amf (2 ×106 cells/ ml) were seeded in 96-well round-bottom plates (Becton Dickinson, NJ). Antibodies were added as indicated and incubated on ice for 1 h. SE pretreated with gangliosides (SAR assays) or native SE (SER-like assays) were then added, mixed by pipetting, centrifuged and incubated overnight on ice. Cells were then resuspended by gentle pipetting, stained with toluidine blue and analysed on slides by light microscopy. Mf with at least three firmly bound erythrocytes were scored as rosette forming cells (RFC). In each experiment, 100 mf from a minimum of three independent wells were
evaluated for RFC formation. Dependency of sialic acid in all assays was examined by treatment of SE and SEG with 0.02 U/ml neuraminidase for 30 min at 37°C (Boehringer Mannheim, Germany).
2.3. Flow cytometry Flow cytometric analysis was performed essentially as described [18]. Briefly, Amf were incubated in HBSS supplemented with 0.3% bovine serum albumin and 0.1% sodium azide for 30 min on ice with optimal dilutions of ED3. Cells were washed twice between stages and further incubated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins (Pharmingen). Subcellular particles and dead and membrane compromised cells were excluded by scatter characteristics and propidium iodide (20 mg/ml) uptake and 104 events were analysed on a FACScan (Becton Dickinson, San Jose, CA) with an argon laser tuned at 488 nm and expression compared to isotype matched mAb controls.
3. Results
3.1. SAR and SER-like acti6ities Between 70 and 90% of Amf bind SE loaded with gangliosides (SEG) leading to rosette formation. Addition of mAb ED3 led to a concentration-dependent inhibition in the percentage of RFC of up to 59% at a concentration of 50 mg/ml. Trypsinized Amf bind between 60 and 70% of native SE resulting in rosette formation (SER-like activity). The mAb ED3 concentration-dependently inhibited up to 48% of SER-like rosette formation (Fig. 1). Treatment of native Amf with IgG2a isotype control (50 mg/ml) or mAb OX42 recognising CD11b/c resulted in no significant differences in RFC numbers compared to controls without antibody (data not shown). Prior treatment of SE or SEG with neuraminidase abolished all rosette formation.
3.2. Expression of Sn by Amf At a saturating ascites dilution of 50 mg/ml, mAb ED3 stained 62% of Amf with a mean channel fluoresence (MCF) of 20.2 (Fig. 2).
4. Discussion SAR expressed by mf have been functionally characterised by different assays. Rat Amf bind SE loaded with gangliosides (SAR activity, [2]) and native SE after prior treatment with proteolytic enzymes such as
K. Frei et al. / Immunology Letters 71 (2000) 167–170
trypsin (SER-like activity, [3,14]). Bone marrow derived murine mf bind native SE in a sialic acid-dependent manner (SER-activity, [5]) through the SER, which was subsequently cloned as the 185 kDa membrane protein Sn [4]. Rat SAR and murine SER activities are temperature independent, not influenced by metabolic inhibitors and in contrast to selectin-family receptors binding sialyl Lewis X [19], are bivalent cation independent [5,20]. SAR and Sn expression is also inverse regulated to major histocompatibility type II (Ia) levels [9,16]. Both SAR and Sn do not bind GM1 and show a specificity for the alpha-anomer of the sialic acid [2,5,15]. IFN gamma inhibits both rat SAR [1] and murine Sn [14,20]
Fig. 1. Influence of monoclonal antibody (mAb) ED3 on sialic acid-dependent receptor (SAR) (shaded) and sheep erythrocyte receptor (SER)-like activity (open bars). Alveolar macrophages (Amf) were incubated with different concentrations of mAb ED3. Data from three experiments show the percentage inhibition of Amf forming rosettes with sheep erythrocytes gangliosides (SEG) or sheep erythrocytes (SE) for each mAb concentration. Experiments with IgG2a isotype control in SAR and SER-like assays in comparable concentrations did not show any inhibitory effect on rosette-formation. Incubation of native Amf with anti-rat CD11b/c (OX42) was also without effect on sialic acid-dependent rosette formation (data not shown).
169
expression although it increases Sn-mRNA in the rat cell line R2 [21]. Regulation of SAR and Sn by granulocyte colony stimulating factor, class I interferons and glucocorticoids is also different [1,17,20]. Although murine Sn activity is demonstrated by peritoneal and resident tissue mf in bone marrow, spleen, lymph nodes and liver, Amf have been considered negative for expression of this molecule. Compared to splenic, peritoneal and bone marrow derived mf, however, rat Amf demonstrate a strong SAR-activity [2]. To clarify the relationship between rat SAR, SERlike activity and Sn expression, a mAb to rat Sn was utilized [14]. Sn expression by rat Amf was demonstrated by FACS analysis and a functional, concentration-dependent inhibition of both SAR and SER-like activities was seen with this antibody which was maximal at dilutions used routinely in FACS analysis. The labelling of 62% of Amf by ED3 in FACS analyses probably represents labelling of a normally distributed population expressing low amounts of Sn with an antibody possessing lower epitope affinity. Alternatively, the epitope recognised by mAb ED3 may be unaccessible on some of the cells. Amf only bind SE in a sialic acid-dependent manner after pre-treatment with trypsin suggesting that either receptor avidity or density is increased following proteolytic treatment [3,14]. This SER-like activity is a less well described phenomenon and was inhibited in our experiments to a lower extent by ED3 compared to SAR activity. These results suggest that this activity is at least in part mediated through Sn expression by rat Amf. On the other hand, higher avidity of the receptor for sialic acid on trypsinized cells may lead to a decrease in steric hindrance of antibody in the inhibition assays. In this study, the expression of Sn is demonstrated by Amf in flow cytometrical analyses with the mAb ED3. Functional inhibition by ED3 in rosetting assays provides evidence that SAR and SER-like activities are mediated by Sn.
Acknowledgements We thank Christine Dijkstra for kindly providing mAb ED3 and K. Baier for technical assistance. This work was supported by a grant from the Hochschuljubila¨umsstiftung der Stadt Wien.
References Fig. 2. Representative flow cytometrical analysis of freshly isolated rat alveolar macrophages (Amf). Data show binding of monoclonal antibody (mAb) ED3 (shaded) in comparison to an isotype control (open).
[1] A. Gessl, G. Boltz-Nitulescu, C. Wiltschke, C. Holzinger, H. Nemt, T. Pernersdorfer, O. Fo¨rster, J. Immunol. 142 (1989) 4372 – 4377.
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K. Frei et al. / Immunology Letters 71 (2000) 167–170
[2] M. Riedl, O. Fo¨rster, H. Rumpold, H. Bernheimer, J. Immunol. 128 (1982) 1205 – 1210. [3] G. Boltz-Nitulescu, B. Ortel, M. Riedl, O. Fo¨rster, Immunology 51 (1984) 177 – 184. [4] P.R. Crocker, S. Kelm, C. Dubois, B. Martin, A.S. McWilliam, D.M. Shotton, J.C. Paulson, S. Gordon, EMBO J. 10 (1991) 1661 – 1669. [5] P.R. Crocker, S. Gordon, J. Exp. Med. 164 (1986) 1862 – 1875. [6] C.D. Dijkstra, E.A. Do¨pp, P. Joling, G. Kraal, Immunology 54 (1985) 589 – 599. [7] A.F. Williams, A.N. Barclay, Ann. Rev. Immunol. 6 (1988) 381 – 405. [8] A.F. Williams, S.J. Davis, Q. He, A.N. Barclay, Cold Spring Harbor Symp. Qant. Bilo 54 (1989) 637–647. [9] I. Stamenkovic, B. Seed, Nature 345 (1990) 74–77. [10] G.L. Wilson, C.H. Fox, A.S. Fauci, J.H. Kehrl, J. Exp. Med. 173 (1991) 137 – 146. [11] G.C. Owens, R.P. Bunge, Glia 3 (1989) 119–128. [12] G. Owens, R.P. Bunge, Neuron 7 (1991) 565–575. [13] B.D. Trapp, Ann. NY Acad. Sci. 605 (1990) 29–43.
.
[14] J.G.M.C. Damoiseaux, E.A. Do¨pp, C.D. Dijkstra, J. Leuk. Biol. 49 (1991) 434 – 441. [15] O. Fo¨rster, G. Boltz-Nitulescu, C. Holzinger, C. Wiltschke, M. Riedl, B. Ortel, A. Fellinger, H. Bernheimer, Mol. Immunol. 23 (1986) 1267 – 1273. [16] E. Travnicek, G. Boltz-Nitulescu, C. Holzinger, O. Fo¨rster, Immunobiology 168 (1984) 260 – 273. [17] J.G.M.C. Damoiseaux, E.A. Do¨pp, R.H.J. Beelen, C.D. Dijkstra, J. Leuk. Biol. 46 (1989) 246 – 253. [18] T. Lucas, W. Krugluger, P. Samorapoompichit, R. Gamperl, H. Beug, O. Fo¨rster, G. Boltz-Nitulescu, FASEB J. 13 (1999) 263 – 272. [19] M.P. Bevilacqua, R.M. Nelson, J. Clin. Invest. 91 (1993) 379– 387. [20] P.R. Crocker, M. Hill, S. Gordon, Immunology 65 (1988) 515– 522. [21] T.K. Van den Berg, I. van Die, C.R. de Lavalette, E.A. Do¨pp, L.D. Smit, P.H. van der Meide, F.J. Tilders, P.R. Crocker, C.D. Dijkstra, J. Immunol. 157 (1996) 3130 – 3138.
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Expression and function of sialoadhesin in rat alveolar macrophages Klemens Frei a,b,*, Christina Steger b, Puchit Samorapoompichit a, Trevor Lucas b, Othmar Fo¨rster b b
a Institute of Histology and Embryology, Uni6ersity of Vienna, Schwarzspanierstraße 17, A-1090 Vienna, Austria Institute of General and Experimental Pathology, Uni6ersity of Vienna, Schwarzspanierstraße 17, A-1090 Vienna, Austria
Received 25 October 1999; received in revised form 5 December 1999; accepted 7 December 1999
Abstract Alveolar macrophages (Amf) represent an immunologically distinct sub-population within the reticuloendothelial system. Phagocytosis and possibly antigen presentation by Amf are essential components of specific and innate primary immune defence processes against inhaled material. The mf-restricted sheep erythrocyte receptor sialoadhesin (Sn) is a member of the immunglobulin superfamily and binds specifically to sialic acid-containing structures such as selectins and was originally identified as the sheep erythrocyte receptor (SER) responsible for sialic acid-dependent binding of native sheep erythrocytes (SE) to resident murine bone marrow macrophages in rosetting assays. Sn expression has been demonstrated on murine and rat mf in lymphatic organs and is recognised by the monoclonal antibody (mAb) ED3 in the rat. In addition, sialic acid-dependent receptor (SAR) activities that mediate rosette formation of alveolar, peritoneal, splenic and bone marrow-resident rat mf with SE pretreated with gangliosides and SER-like activities between native SE and trypsinised Amf, have been described. The binding activities of both SAR and Sn show similar characteristics suggesting that these molecules are closely structurally related or identical. To clarify the relationship between Sn, SAR and SER-like activities, the binding of mAb ED3 to isolated rat Amf was investigated by flow cytometry and rosetting assays. It is demonstrated that rat Amf express Sn and evidence is provided that SAR and SER-like activities are mediated by Sn. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Sialic acid-dependent recptor (SAR); Sialoadhesin (Sn); Immunglobulin superfamily; ED3; Rosette assay
1. Introduction Sialic acid-dependent receptor (SAR) binds sialic acid-containing structures and is responsible for cellular interactions between rat mf (alveolar, peritoneal, splenic and bone marrow) and sheep erythrocytes (SE) pretreated with gangliosides (SEG) [1,2]. Treatment of SE with neuraminidase as well as addition of sialic acid-containing oligosaccharides inhibits SAR-activity [3]. A SAR called sialoadhesin (Sn) was initially characterized from murine resident bone marrow mf binding of native SE which is inhibited by pretreatment with neuraminidase or gangliosides [4]. Sn expression has * Corresponding author. Tel.: +43-1-427761346; fax: +43-142779613. E-mail address: [email protected] (K. Frei)
been demonstrated on murine mf in lymphatic organs and Kupffer cells [5]. The distribution of this receptor in lymphatic organs of the rat has also been characterised by the monoclonal antibody (mAb) ED3 [6]. Cloning of murine Sn demonstrated this receptor as a new member of the immunglobulin superfamily [7,8] with 17 immunoglobulin-like domains closely related to CD22 [9,10] and myelin-associated glycoprotein [11– 13]. Binding activities of both SAR and Sn is independent of divalent cations [2,5,14], show similar pH characteristics [2,5] and affinity for different gangliosides [2,4,15]. SAR and Sn activities are also not inhibited by metabolic inhibitors and do not trigger phagocytosis [5,12]. The expression of these receptors has been inversely correlated to major histocompatibility class II (Ia) levels [5,16] and is similarly regulated by hormones and cytokines [1,5,17]. These observations led to the
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K. Frei et al. / Immunology Letters 71 (2000) 167–170
conclusion that both receptors show a close functional relationship or identity. The aim of the present study was to investigate the expression and function of Sn on rat Amf. The expression of Sn was demonstrated by flow cytometrical analyses with the mAb ED3. Functional inhibition by this antibody in rosetting assays provides evidence that SAR and SER-like activities are mediated by Sn.
2. Materials and methods
2.1. Cell preparation Amf were isolated from 150 to 160 g male Louvain rats (Himberg, Austria). Amf were harvested by bronchoalveolar lavage in Hank’s buffered saline solution (HBSS buffer) supplemented with 0.25% EDTA and 2% fetal calf serum (Flow laboratories, Irvine, Scotland). Cell viabilities were estimated by trypan blue exclusion and purity of Amf populations was routinely assessed as \ 99% by morphology and non-specific esterase staining as described previously. For SER-like assays, Amf were resuspended in a 0.05% trypsin solution (Gibco BRL, UK) and incubated for 15 min at 37°C. After washing in HBSS, cells were resuspended in hepes-buffered RPMI 1640 (Gibco BRL, UK). Erythrocytes from male sheep (Immuno, Austria) stored in Alserver’s solution at 4°C for up to 5 days, were incubated for 1 h at 37°C in a 100 mg/ml solution of bovine brain gangliosides (Sigma, St. Louis, MO), primarily containing GM1 and GD1a, pretreated in an ultrasound device.
2.2. SAR and SER-like assays The mouse mAb ED3 recognising rat Sn was generously provided by Christine Dijkstra, Free University of Amsterdam, Holland. Murine IgG2a isotype control mAb was purchased from DAKO (Glostrup, Denmark). Purified mAb OX42 directed against rat CD11b/ c was purchased from Pharmingen (San Diego, CA). All antibodies were absorbed with SE to remove residual binding activity. A total of 50 ml suspensions of Amf (2 ×106 cells/ ml) were seeded in 96-well round-bottom plates (Becton Dickinson, NJ). Antibodies were added as indicated and incubated on ice for 1 h. SE pretreated with gangliosides (SAR assays) or native SE (SER-like assays) were then added, mixed by pipetting, centrifuged and incubated overnight on ice. Cells were then resuspended by gentle pipetting, stained with toluidine blue and analysed on slides by light microscopy. Mf with at least three firmly bound erythrocytes were scored as rosette forming cells (RFC). In each experiment, 100 mf from a minimum of three independent wells were
evaluated for RFC formation. Dependency of sialic acid in all assays was examined by treatment of SE and SEG with 0.02 U/ml neuraminidase for 30 min at 37°C (Boehringer Mannheim, Germany).
2.3. Flow cytometry Flow cytometric analysis was performed essentially as described [18]. Briefly, Amf were incubated in HBSS supplemented with 0.3% bovine serum albumin and 0.1% sodium azide for 30 min on ice with optimal dilutions of ED3. Cells were washed twice between stages and further incubated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins (Pharmingen). Subcellular particles and dead and membrane compromised cells were excluded by scatter characteristics and propidium iodide (20 mg/ml) uptake and 104 events were analysed on a FACScan (Becton Dickinson, San Jose, CA) with an argon laser tuned at 488 nm and expression compared to isotype matched mAb controls.
3. Results
3.1. SAR and SER-like acti6ities Between 70 and 90% of Amf bind SE loaded with gangliosides (SEG) leading to rosette formation. Addition of mAb ED3 led to a concentration-dependent inhibition in the percentage of RFC of up to 59% at a concentration of 50 mg/ml. Trypsinized Amf bind between 60 and 70% of native SE resulting in rosette formation (SER-like activity). The mAb ED3 concentration-dependently inhibited up to 48% of SER-like rosette formation (Fig. 1). Treatment of native Amf with IgG2a isotype control (50 mg/ml) or mAb OX42 recognising CD11b/c resulted in no significant differences in RFC numbers compared to controls without antibody (data not shown). Prior treatment of SE or SEG with neuraminidase abolished all rosette formation.
3.2. Expression of Sn by Amf At a saturating ascites dilution of 50 mg/ml, mAb ED3 stained 62% of Amf with a mean channel fluoresence (MCF) of 20.2 (Fig. 2).
4. Discussion SAR expressed by mf have been functionally characterised by different assays. Rat Amf bind SE loaded with gangliosides (SAR activity, [2]) and native SE after prior treatment with proteolytic enzymes such as
K. Frei et al. / Immunology Letters 71 (2000) 167–170
trypsin (SER-like activity, [3,14]). Bone marrow derived murine mf bind native SE in a sialic acid-dependent manner (SER-activity, [5]) through the SER, which was subsequently cloned as the 185 kDa membrane protein Sn [4]. Rat SAR and murine SER activities are temperature independent, not influenced by metabolic inhibitors and in contrast to selectin-family receptors binding sialyl Lewis X [19], are bivalent cation independent [5,20]. SAR and Sn expression is also inverse regulated to major histocompatibility type II (Ia) levels [9,16]. Both SAR and Sn do not bind GM1 and show a specificity for the alpha-anomer of the sialic acid [2,5,15]. IFN gamma inhibits both rat SAR [1] and murine Sn [14,20]
Fig. 1. Influence of monoclonal antibody (mAb) ED3 on sialic acid-dependent receptor (SAR) (shaded) and sheep erythrocyte receptor (SER)-like activity (open bars). Alveolar macrophages (Amf) were incubated with different concentrations of mAb ED3. Data from three experiments show the percentage inhibition of Amf forming rosettes with sheep erythrocytes gangliosides (SEG) or sheep erythrocytes (SE) for each mAb concentration. Experiments with IgG2a isotype control in SAR and SER-like assays in comparable concentrations did not show any inhibitory effect on rosette-formation. Incubation of native Amf with anti-rat CD11b/c (OX42) was also without effect on sialic acid-dependent rosette formation (data not shown).
169
expression although it increases Sn-mRNA in the rat cell line R2 [21]. Regulation of SAR and Sn by granulocyte colony stimulating factor, class I interferons and glucocorticoids is also different [1,17,20]. Although murine Sn activity is demonstrated by peritoneal and resident tissue mf in bone marrow, spleen, lymph nodes and liver, Amf have been considered negative for expression of this molecule. Compared to splenic, peritoneal and bone marrow derived mf, however, rat Amf demonstrate a strong SAR-activity [2]. To clarify the relationship between rat SAR, SERlike activity and Sn expression, a mAb to rat Sn was utilized [14]. Sn expression by rat Amf was demonstrated by FACS analysis and a functional, concentration-dependent inhibition of both SAR and SER-like activities was seen with this antibody which was maximal at dilutions used routinely in FACS analysis. The labelling of 62% of Amf by ED3 in FACS analyses probably represents labelling of a normally distributed population expressing low amounts of Sn with an antibody possessing lower epitope affinity. Alternatively, the epitope recognised by mAb ED3 may be unaccessible on some of the cells. Amf only bind SE in a sialic acid-dependent manner after pre-treatment with trypsin suggesting that either receptor avidity or density is increased following proteolytic treatment [3,14]. This SER-like activity is a less well described phenomenon and was inhibited in our experiments to a lower extent by ED3 compared to SAR activity. These results suggest that this activity is at least in part mediated through Sn expression by rat Amf. On the other hand, higher avidity of the receptor for sialic acid on trypsinized cells may lead to a decrease in steric hindrance of antibody in the inhibition assays. In this study, the expression of Sn is demonstrated by Amf in flow cytometrical analyses with the mAb ED3. Functional inhibition by ED3 in rosetting assays provides evidence that SAR and SER-like activities are mediated by Sn.
Acknowledgements We thank Christine Dijkstra for kindly providing mAb ED3 and K. Baier for technical assistance. This work was supported by a grant from the Hochschuljubila¨umsstiftung der Stadt Wien.
References Fig. 2. Representative flow cytometrical analysis of freshly isolated rat alveolar macrophages (Amf). Data show binding of monoclonal antibody (mAb) ED3 (shaded) in comparison to an isotype control (open).
[1] A. Gessl, G. Boltz-Nitulescu, C. Wiltschke, C. Holzinger, H. Nemt, T. Pernersdorfer, O. Fo¨rster, J. Immunol. 142 (1989) 4372 – 4377.
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K. Frei et al. / Immunology Letters 71 (2000) 167–170
[2] M. Riedl, O. Fo¨rster, H. Rumpold, H. Bernheimer, J. Immunol. 128 (1982) 1205 – 1210. [3] G. Boltz-Nitulescu, B. Ortel, M. Riedl, O. Fo¨rster, Immunology 51 (1984) 177 – 184. [4] P.R. Crocker, S. Kelm, C. Dubois, B. Martin, A.S. McWilliam, D.M. Shotton, J.C. Paulson, S. Gordon, EMBO J. 10 (1991) 1661 – 1669. [5] P.R. Crocker, S. Gordon, J. Exp. Med. 164 (1986) 1862 – 1875. [6] C.D. Dijkstra, E.A. Do¨pp, P. Joling, G. Kraal, Immunology 54 (1985) 589 – 599. [7] A.F. Williams, A.N. Barclay, Ann. Rev. Immunol. 6 (1988) 381 – 405. [8] A.F. Williams, S.J. Davis, Q. He, A.N. Barclay, Cold Spring Harbor Symp. Qant. Bilo 54 (1989) 637–647. [9] I. Stamenkovic, B. Seed, Nature 345 (1990) 74–77. [10] G.L. Wilson, C.H. Fox, A.S. Fauci, J.H. Kehrl, J. Exp. Med. 173 (1991) 137 – 146. [11] G.C. Owens, R.P. Bunge, Glia 3 (1989) 119–128. [12] G. Owens, R.P. Bunge, Neuron 7 (1991) 565–575. [13] B.D. Trapp, Ann. NY Acad. Sci. 605 (1990) 29–43.
.
[14] J.G.M.C. Damoiseaux, E.A. Do¨pp, C.D. Dijkstra, J. Leuk. Biol. 49 (1991) 434 – 441. [15] O. Fo¨rster, G. Boltz-Nitulescu, C. Holzinger, C. Wiltschke, M. Riedl, B. Ortel, A. Fellinger, H. Bernheimer, Mol. Immunol. 23 (1986) 1267 – 1273. [16] E. Travnicek, G. Boltz-Nitulescu, C. Holzinger, O. Fo¨rster, Immunobiology 168 (1984) 260 – 273. [17] J.G.M.C. Damoiseaux, E.A. Do¨pp, R.H.J. Beelen, C.D. Dijkstra, J. Leuk. Biol. 46 (1989) 246 – 253. [18] T. Lucas, W. Krugluger, P. Samorapoompichit, R. Gamperl, H. Beug, O. Fo¨rster, G. Boltz-Nitulescu, FASEB J. 13 (1999) 263 – 272. [19] M.P. Bevilacqua, R.M. Nelson, J. Clin. Invest. 91 (1993) 379– 387. [20] P.R. Crocker, M. Hill, S. Gordon, Immunology 65 (1988) 515– 522. [21] T.K. Van den Berg, I. van Die, C.R. de Lavalette, E.A. Do¨pp, L.D. Smit, P.H. van der Meide, F.J. Tilders, P.R. Crocker, C.D. Dijkstra, J. Immunol. 157 (1996) 3130 – 3138.