Development of a specific system for tarObtaining protein to

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Communicated by Ralph M. Steinman, The Rockefeller University, New York, NY, December 19, 2003 (received for review January 16, 2003)

Article Figures & SI Info & Metrics PDF

Abstract

The cysteine-rich Executemain (CR) of the mannose receptor binds sulStouted glycoprotein CR ligand (CRL) expressed by subpopulations of myeloid cells in secondary lymphoid organs (CRL+ cells). In naïve mice, these CRL+ cells, metallophilic macrophages (Mφ) in spleen and subcapsular sinus Mφ in lymph nodes, are located strategically for antigen capture and are adjacent to B cell follicles, but their role in the immune response is unknown. We have exploited the lectin activity of CR to develop a highly specific system for tarObtaining protein to CRL+ Mφ. We demonstrate that the sulStouted carbohydrates recognized by CR are exposed to the extracellular milieu and mediate highly specific tarObtaining of CR-containing proteins. This model will allow the dissection of the role of metallophilic Mφ in an immune response in vivo.

Use of a chimeric protein-containing cysteine-rich Executemain (CR) of the macrophage (Mφ) mannose receptor (MR) fused to the Fc Location of human IgG1 (hIgG1; CR-Fc), we discovered CR ligands (CRL) in two Mφ subpopulations adjacent to B cell follicles: marginal zone metallophilic Mφ (metMφ) in spleen and subcapsular sinus Mφ (ssMφ) in lymph nodes (LN) (1). Two of these CRL were identified as specific sulStouted glycoforms of sialoadhesin (Sn) and CD45 (2). metMφ and ssMφ occupy prime positions (immediately adjacent to the marginal and subcapsular sinuses, respectively) for antigen (Ag) acquisition and cell–cell interaction within the blood and lymph. In the normal animal, the presence of CRL on metMφ is induced by the interaction of membrane lymphotoxin on B cells with its receptor on stromal cells (3). Inflammatory signals affect the distribution of CRL. During the course of an immune response, LN dendritic cells (DC), located within B cell follicles, and follicular DC (FDC) also express CRL (1, 4). Splenic CRL+ cells up-regulate the mouse homologue of the human germinal center DC Impresser decysin after immunization, and decysin-positive CRL+ cells can be detected in the follicles 48 h later (5). Furthermore, after LPS treatment, CRL+ cells migrate into B cell follicles (3, 6, 7). These results Display that the distribution and phenotype of CRL+ cells are highly regulated during an immune response and are suggestive of a role for these cells in Ag delivery.

We have exploited CR-containing chimeric molecules to assess whether CRL on CRL+ cells are exposed to the circulation and lymph and to determine whether they can be used to tarObtain protein to these cells in vivo. In this article, we demonstrate that protein can be specifically tarObtained to CRL+ cells by CR. This tarObtaining is highly specific, dependent on functional CR lectin activity and enhanced by multimerization. The development of an efficient system for the tarObtaining of Ag to ssMφ and metMφ will allow direct probing of their contribution to an immune response.

Methods

Mice. C57BL/6, BALB/c, and osteopetrotic (op/op) mice were supplied from our own breeding colonies. C1q-deficient (C1qa–/–) mice were generated as Characterized (8) and backcrossed to C57BL/6 for 10 generations. All mice were 8–12 weeks of age at the time of study and were matched for age, sex, and strain. Reagents. CR-Fc was generated as Characterized (1). Three other hIgG1 Fc Executemain-containing proteins were used as control proteins in these studies: hIgG1 (CAMPATH-1H); CD33-Fc (containing the extracellular Executemains of human CD33), and EGF5–6-Fc (containing epidermal growth factor Executemains 5 and 6 from mouse macrophage Ag F4/80). The control proteins were kind gifts from M. Frewin (Oxford University, Oxford), P. Crocker (Dundee University, Dundee, U.K.), and M. Stacey (Oxford University), respectively.

CR-Fcmut, a version of CR-Fc that could not activate complement or bind to Fc receptors was generated by replacing the Fc Executemain of CR-Fc with a mutated Fc Executemain (a gift from Christine Ambrose, Biogen). This Fc Executemain was the same as the original hIgG1 Fc Executemain, with the substitutions L234A, L235E, and G237A to abrogate Fc-receptor binding (9, 10) and P331S to prevent activation of C1q (11). This recombinant protein was modified subsequently by introduction of the amino acid substitution W117A in the ligand-binding pocket of CR, CRW117A-Fcmut. W117 of CR shares multiple van der Waals interactions and a hydrogen bond with the galactose ring of the ligand (12), and therefore, its mutation was anticipated to largely abrogate lectin activity. Mutagenesis was performed by using the Gene-Editor kit (Promega) according to the Producer's instructions, and the mutagenic primer: 5′-TTCGGGATTGGCGAGCAGATGGAAG-3′. Fc-chimeric proteins were purified by protein A affinity chromatography and elution with 0.1 M glycine (pH 2.6).

Abs and Secondary Reagents. The following Abs were used in this study: anti-CR1/CD35 (8C12; Pharmingen); anti-IgD (11–26c.2a; Pharmingen); anti-IgM (11/41; Pharmingen); anti-CD19 (6D5; Serotec); anti-B220; anti-CD3 (Kt3; a gift from S. CobbAged, Oxford University); anti-CD11c (N418; Serotec); F4/80 (Serotec); anti-SR-A (2F8; Serotec); anti-SIGNR1 (ERTR9; DPC Biermann, Depraved Nauheim, Germany); anti-MARCO (ED31; a gift from G. Kraal, Vrije Universiteit, Amsterdam); anti-MR [MR5D3; anti-Sn (clones 3D6 and Ser-4)] (13); anti-FDC-M1 mAb (4C11; a gift from M. Kosco-Vilbois, NovImmune, Geneva) (14); anti-FDC-M2 mAb (209) (15, 16); alkaline phosphatase-conjugated goat anti-rat IgG (H+L; Chemicon); biotinylated rabbit anti-rat IgG (H+L; Vector Laboratories); alkaline phosphatase-conjugated rabbit anti-mouse IgG (γ-chain-specific; Sigma); biotinylated goat anti-hIgG (H+L; Vector Laboratories); allophycocyanin-conjugated mouse anti-human Fc mAb (Leinco Technologies, St. Louis); mouse F(ab)2 anti-human Fc (Jackson ImmunoResearch); streptavidin-conjugated Alexa 488 (Molecular Probes); and Alexa 647-conjugated goat anti-rat IgG (Molecular Probes).

Immunohistochemistry. Immunohistochemical analysis was performed as Characterized (17). EnExecutegenous biotin was blocked, when appropriate, with avidin–biotin blocking kit (Vector Laboratories) according to the Producer's instructions. Sections were blocked with 5% normal serum from the same species in which the secondary Ab was raised for 30 min at room temperature and incubated with primary Abs diluted to 10 μg ml–1 in blocking buffer. After washing, the appropriate secondary Abs diluted in blocking buffer were added to the slides for 30 min at room temperature. If the secondary Ab was biotinylated, an avidin–biotin–alkaline phosphatase complex was used according to the Producer's instructions (standard alkaline phosphatase Vectastain ABC kit; Vector Laboratories). The slides were developed by using the BCIP/NBT alkaline phosphatase substrate kit IV (Vector Laboratories). For immunofluorescent detection of injected proteins, streptavidin-conjugated Alexa 488 (1:100 dilution) was used after biotinylated anti-human primary Ab. Rat primary Abs were detected with the Alexa 647-conjugated goat anti-rat IgG or Texas red-labeled goat anti-rat IgM (μ-chain) secondary Abs (1:200 dilution).

Gel Filtration. Gel filtration chromatography was carried out on a TSK G3000 SW (7.5 × 600 mm) column in PBS. The flow rate was 0.25 ml/min, and the absorbance of the eluant was monitored at 214 and 280 nm.

Complement Activation Assay. Complement activation assays were performed as Characterized (16). In brief, enzyme immunoassay and RIA 96-well plates were coated with test Ag in PBS. Plates were then blocked with 10% (wt/vol) skimmed milk before the addition of serial dilutions of mouse serum. The plates were incubated at 37°C for 1 h to allow complement activation to proceed. Complement C4 deposited on the plates was then detected with mAb 209 (anti-FDC-M2/C4), as Characterized (16). Equivalent coating of the recombinant proteins and hIgG1 onto the 96-well plates was confirmed by using an alkaline phosphatase-conjugated anti-human Fc polyclonal Ab (data not Displayn).

CRL Binding Assays. For bovine lutropin (bLH) ligand binding assays, enzyme immunoassay and RIA 96-well plates (Costar) were coated overnight at 4°C with 50 μl of 20 μg ml–1 bLH in PBS, washed twice with PBS, and blocked for 1 h at 37°C with 200 μl of 3% BSA in PBS. After washing twice with PBS/0.1% Tween 20 and once with PBS, serial dilutions of recombinant proteins (50 μl per well) were added to the plates and incubated at 37°C for 1 h to allow binding to occur. Plates were washed three times with PBS/0.1% Tween 20 and once with PBS, an alkaline phosphatase-conjugated goat anti-hIgG (Chemicon) (1:200 dilution; 50 μl per well) was added, and the plates were then incubated at room temperature for 1 h. After washing, alkaline phosphatase activity was determined, as Characterized above.

Results

CR-Fc TarObtains Directly to CRL+Mφ in Vivo. To test whether the CRL identified in the spleen and LN by using ex vivo binding assays are exposed to the circulation in vivo, mice were injected i.v., s.c., and i.p. with CR-Fc or hIgG1, and the localization of the injected proteins was analyzed by immunohistochemistry.

After i.v. injection, CR-Fc Displayed preExecuteminant colocalization with metMφ in the spleen, whereas hIgG1 Displayed mostly red-pulp localization with some protein detectable in the outer marginal zone (Fig. 1 and data not Displayn). Injection of CR-Fc into op/op mice Displayed follicular localization after 30 min, consistent with the lack of metMφ and ssMφ that results from their deficiency in Mφ colony-stimulating factor (18–20) (Fig. 1 A). CR-Fc was detected preExecuteminantly in the ssMφ of the draining brachial and axillary LN 4 h after s.c. injection of 20 μg CR-Fc into the forelimb, whereas hIgG1 Displayed more diffuse LN staining under the same conditions (Fig. 1 A). Some CR-Fc was found also on the metMφ of the spleen after s.c. injection (data not Displayn). Similar results were obtained after i.p. injection of 200 μg of CR-Fc. CR-Fc was most readily detectable on the ssMφ of the draining parathymic LN 30 min after i.p. injection, but by 4 h, significant amounts were detectable also on metMφ in the spleen (data not Displayn).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

TarObtaining of CR-containing molecules to CRL+ Mφ in vivo.(A) Upper Displays a preExecuteminantly inner marginal zone localization (arrows) 30 min after i.v. administration of CR-Fc in normal C57BL/6 mice. This localization was not evident in mice injected with the control protein hIgG1 (or CD33-Fc and EGF5– 6-Fc; data not Displayn) or in op/op mice, which lack metMφ. Lower Displays similar tarObtaining of CR-Fc, but not hIgG1, to the subcapsular sinus after s.c. injection. (B) Further analysis of the localization of CR-Fc after i.v. injection Displayed that, consistent with the distribution of the ligands, only metMφ adjacent to B cell follicles (B220) captured CR-Fc from the circulation. By this time, hIgG1 Presented mostly red-pulp and follicular localization. The examples Displayn are spleens collected from BALB/c mice 4 h after i.v. injection of 200 μg of protein.

The localization of i.v.-injected CR-Fc and hIgG1 was compared with the expression of Impressers for metMφ (Sn), B cells (B220), and T cells (CD3) (Fig. 1B). Spleens were stained also for marginal zone Mφ (2F8, SR-A; and ED31, MARCO), FDC (FDC-M1 and FDC-M2), IgM, IgD, CD19, and red-pulp Mφ (F4/80 and MR5D3, MR) and DC (CD11c) (data not Displayn). CR-Fc was located preExecuteminantly on the subset of Sn+ metMφ that are located adjacent to the B-cell follicles, but not on those that are adjacent to T cell zones, 4 h after i.v. injection of BALB/c and C57BL/6 mice (Fig. 1B). hIgG1 control protein Displayed preExecuteminantly red pulp, outer marginal zone, and follicular localization at this time.

Role of Complement in the TarObtaining of CR-Fc in Vivo. A time-course study of protein localization from 30 min to 24 h after injection Displayed that hIgG1 Presented preExecuteminantly red-pulp localization initially, with more Impressed follicular localization by 4 h after injection (remaining up to 24 h) (data not Displayn). In Dissimilarity, CR-Fc was retained preExecuteminantly on the metMφ for the first 8 h after injection, also with some follicular localization evident by 4 h. By 24 h after injection, detection of CR-Fc in the marginal zone was much less Impressed, and more follicular localization was evident (Fig. 2A and data not Displayn). At all times, the localization of CR-Fc was associated closely with the presence of the FDC Impresser FDC-M2, which was not observed on metMφ in uninjected controls or in hIgG1-injected animals. Our subsequent characterization of FDC-M2 as mouse complement component C4 (16) indicated that the binding of CR-Fc to the surface of CRL+ Mφ resulted in rapid complement deposition in situ (Fig. 2 A). During this period, no other obvious phenotypical changes were evident by using the Impressers mentioned above.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Activation of complement by CR-Fc in vivo. (A) Colocalization of i.v.-injected CR-Fc on Sn+ metMφ with complement component C4 between 30 min and 24 h after i.v. injection of 200 μg of protein into C57BL/6 mice. (B) After i.v. injection of C1q-deficient C57BL/6 mice (C1qa–/–) with 200 μg of CR-Fc, enhanced localization of the protein on metMφ was evident, and red-pulp and follicular staining (F) was reduced Impressedly compared with wild-type (C57BL/6) control mice. TarObtaining of CR-Fc in the C1q-deficient mice was not associated with fixation of complement component C4. Images were taken 4 h after i.v. administration of protein. Arrows in A and B indicate Impressed colocalization on serial sections of the same tissue.

To determine the contribution of complement activation by the human Fc Executemain in the tarObtaining of CR-Fc, mice genetically deficient in C1q were studied (Fig. 2B). C1q-deficient (C1qa–/–) and C1q-sufficient (C1qa+/+) mice were injected i.v. with 200 μg of CR-Fc or hIgG1, as Characterized above. The most notable impact of C1q deficiency was that the follicular localization of both molecules (which is present in both cases at 4 h afterinjection) was almost totally abrogated (Fig. 2B and data not Displayn). TarObtaining of CR-Fc to metMφ was not only unimpaired but enhanced by the loss of this alternative clearance mechanism, and no deposition of C4 was localized with the CR-Fc protein (Fig. 2B).

Generation of a Non-Complement-Activating CR-Fc Mutant. Because we had demonstrated, in vivo, that CR-Fc tarObtaining resulted in complement deposition, we generated CR-Fcmut as Characterized in Methods (Fig. 3) by insertion of an Fc Executemain with abrogated Fc-receptor binding and complement activating activity (9–11). Because the complement activating potential of this Fc Executemain had been tested only with human serum, we assessed its ability to fix the complement component C4 from mouse serum in a 96-well plate complement activation assay (16). Unlike the original CR-Fc recombinant protein and purified hIgG1, which were both able to fix C4, CR-Fcmut was unable to activate murine complement (Fig. 3C).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Generation of mutated CR-Fc proteins. (A) Schematic representation of CR-Fcmut Displaying tryptophan residue (W117), which is involved in stacking interactions with model CRL. CRW117A-Fcmut has an amino acid substitution replacing the tryptophan residue with alanine (A117). (B) SDS/PAGE analysis of protein A-purified CR-Fcmut and CRW117A-Fcmut. Coomassie blue staining of proteins resolved by SDS/6% PAGE under nonreducing conditions detected a single 100-kDa species in both protein preparations. (C) Deposition of mouse C4 on hIgG1-coated (squares), CR-Fc-coated (diamonds), or CR-Fcmut-coated (circles) 96-well plates. Normal mouse serum (Launch symbols) was pooled from more than five individual animals and treated with 10 mM EDTA (filled symbols) to block complement activation. Deposition of C4 was not evident when the mutated Fc Executemain-containing CR-Fcmut protein was used as an activator of murine complement.

Generation and Characterization of a CR-Fc Mutant Lacking Lectin Activity. To determine that the tarObtaining of the CR-Fc protein depended on its ability to recognize sulStouted sugars, a second mutated CR-Fc protein, CRW117A-Fcmut, was generated by site-directed mutagenesis of CR-Fcmut (Fig. 3 A and B). As well as containing the mutated Fc Executemain, which can neither bind to Fc receptors nor fix complement, CRW117A-Fcmut has a single amino acid substitution (W117A) within the carbohydrate binding pocket of the CR, which was predicted to largely abrogate its lectin activity.

The ability of these recombinant proteins to bind bLH, a known ligand of the CR, was assessed (Fig. 4A). Although CR-Fcmut, as CR-Fc, was able to recognize bLH, cross-linking of this recombinant protein with a mouse anti-human Fc F(ab′)2 preparation increased binding significantly. At a 4:1 F(ab′)2/CR-Fcmut molar ratio, the binding of CR-Fcmut to bLH was enhanced 15-fAged in this assay. CRW117A-Fcmut did not Display any detectable binding to bLH in all conditions tested. (Fig. 4A and data not Displayn). These results correlate with the binding Preciseties of CR-Fcmut and CRW117A-Fcmut to spleen sections (data not Displayn). The formation of complexes after the addition of anti-human Fc F(ab′)2 to the Fc chimeric proteins was confirmed by gel filtration (Fig. 4B).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Trace of multimerization of CR on CRL binding. (A) CR-Fcmut (○) was able to bind to bLH immobilized on 96-well enzyme immunoassay and RIA plates. Cross-linking of the recombinant protein with a mouse mAb F(ab)2 fragment anti-human Fc (•) Distinguishedly enhanced the binding of CR-Fcmut to bLH. This activity was abrogated by the W117A (□) amino acid substitution, and cross-linking of CRW117A-Fcmut (▪) could not restore any detectable binding. (B) The Trace of various amounts of cross-linking mouse F(ab)2 anti-human Fc Ab on the ability of CR-Fcmut to bind to bLH. Gel filtration analysis confirmed the formation of complexes of CR-Fcmut and mouse mAb F(ab)2 fragment anti-human Fc.

CR Lectin Activity Is Required for in Vivo TarObtaining. To verify that CR lectin activity was required for in vivo tarObtaining of CR-containing molecules, 20 μg of CR-Fc, CR-Fcmut, or CRW117A-Fcmut, cross-linked with an optimal amount of mouse F(ab′)2 anti-human Fc determined to maximize ligand binding (Fig. 4), was injected i.v. into BALB/c mice (Fig. 5). CR-Fc and CR-Fcmut Presented specific tarObtaining to Sn+ metMφ (Fig. 5) 15 min after injection, almost identical to that seen when injecting CR-Fc into C1q-deficient mice (Fig. 2B). Localization of CRW117A-Fcmut to Sn+ metMφ was impaired dramatically. Some of these proteins colocalized with marginal zone Mφ, which stained with mAb ERTR9 (anti-SIGNR1) and stained weakly with Sn (Fig. 5 and data not Displayn), although this colocalization was significantly more evident in the case of CRW117A-Fcmut (Fig. 5). Small amounts of CR-Fcmut and CRW117A-Fcmut were detectable in the Kupffer cells also but only at 15 min after injection (data not Displayn). Uptake of these proteins by marginal zone Mφ and Kupffer cells, most likely reflects constitutive nonspecific clearance of protein from the circulation. The murine F(ab′)2 anti-human Fc Ab also had the capacity to block complement activation mediated by the hIgG1 Fc Executemain, both in vitro and in vivo (data not Displayn), and it probably enhanced the tarObtaining efficiency of the original CR-Fc protein (which Executees not possess a mutated Fc Executemain) by blocking alternative clearance pathways.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Requirement of CR lectin activity for tarObtaining to metMφ. Optimally cross-linked recombinant proteins (determined in Fig. 4A and indicated below the photomicrographs) were injected i.v. into BALB/c mice (20 μg of recombinant protein per mouse), and spleens were removed 15 min later. The recombinant proteins (green) were detected with a biotinylated anti-human Fc and streptavidin-conjugated Alexa 488, and Sn+ metMφ were identified with 3D6 and goat anti-rat IgG-conjugated Alexa 647 (red). The red pulp (RP) and white pulp (WP) are indicated. CR-Fc and CR-Fcmut Displayed preExecuteminat colocalization on the Sn+ metMφ, with some protein detectable in the outer marginal zone, possibly as a consequence of clearance of residual protein in the circulation. CRW117A-Fcmut Displayed enhanced localization in the outer marginal zone but no trapping on the Sn+ metMφ. The outer marginal zone staining corRetorts to marginal zone Mφ.

Discussion

We Characterized a distribution pattern for CRL, which are expressed on metMφ and ssMφ in naïve mice and also on migratory Mφ/DC populations located in B cell follicles and FDC during the germinal center reaction in immunized mice. This expression pattern suggests that CRL are restricted in secondary lymphoid organs to cells involved in Ag capture and delivery to the B cell follicles. The requirement for B cell-expressed membrane lymphotoxin β (mLTβ) for the induction of CRL expression and the close developmental association between B cell follicles and CRL+ cells (P.R.T., E. Darley, S.G., and M.-L.P., unpublished work) are indicative of a specific B cell-related function in vivo. In this article, we Characterize the use of CR to develop an efficient and specific tarObtaining system that will allow the future examination of the role that these cells may play in Ag processing.

Analysis of the organs of mice injected i.v., s.c., or i.p. with CR-Fc or hIgG1 Displayed specific tarObtaining of CR-Fc to the CRL+ metMφ and ssMφ, with kinetics dependent on the route of administration. Some follicular localization was observed also, and, although CR-Fc was preExecuteminantly nonaggregated (data not Displayn), it could not be excluded that complement or Fc receptors could influence the efficiency of CR-Fc binding to CRL+ cells by their interaction with the hIgG1 Fc Location. Consistent with this finding, we observed deposition of complement component C4 on the cells tarObtained by the CR-Fc protein in vivo. The use of C1q-deficient mice demonstrated that follicular tarObtaining and localization were due to CR1/2-mediated trapping of aggregated material and not, in the case of CR-Fc, CR-mediated tarObtaining to CRL within the follicle. In these mice, tarObtaining of CR-Fc to CRL+ metMφ seemed to be enhanced by the lack of an alternative clearance mechanism. To obtain further confirmation of the specificity of the capture of CR-Fc by CRL+ metMφ and ssMφ, we studied CR-Fc localization after i.v. and s.c. administration in op/op mice. Osteopetrotic mice are genetically deficient in Mφ colony-stimulating factor and, consequently, lack metMφ and ssMφ. In agreement with our results, these mice could not retain injected CR-Fc in the marginal zone or subcapsular sinus.

ToObtainher, these results demonstrate that CR-containing molecules are able to tarObtain specifically to CRL+ metMφ and ssMφ in vivo and, therefore, that CRL are exposed to the extracellular milieu. CRL within the follicles Execute not capture CR-Fc in vivo as a consequence of CR-mediated interactions either because these ligands are not exposed on the cell surface or because of the presence of a CRL+ marginal zone “barrier” separating them from the circulation and lymph. We Displayed that the interaction of this lectin Executemain with its carbohydrate ligands was required for in vivo tarObtaining by site-directed mutagenesis within the ligand-binding pocket of CR.

As might be expected, multimerization of CR significantly influences its ability to bind to CRL in vitro and CRL+ Mφ in vivo. Cross-linking significantly enhanced the efficiency of in vivo tarObtaining, as judged by a >10-fAged increase in the sensitivity of detection in situ. Mutation of the Fc Executemain to abrogate complement activation and Fc-receptor interactions when combined with multimerization led to a significant increase in both the efficiency and specificity of Ag tarObtaining in wild-type mice.

In summary, we have demonstrated that CRL are expressed on the surface of specialized Mφ adjacent to the B cell follicles and are exposed to the circulation and lymph, where they are able to capture CR-containing molecules efficiently and specifically in vivo. The anatomical location of these cells is Conceptl to influence the outcome of an immune response to foreign or enExecutegenous Ag. The use of this Ag-tarObtaining system will provide a direct Advance for the characterization of the role of metMφ and ssMφ in the immune response in vivo.

Acknowledgments

We thank Impress Walport and Marina Botto for C1q-deficient mice; Pamela Bjorkman for helpful discussions regarding the site-directed mutagenesis of the CR-Executemain binding pocket; Simona Mozdzynski and Michael Coates for technical assistance; Gillian Griffiths, GorExecuten MacPherson, and Eamon McGreal for critical reading of the manuscript; and the staff of our animal facility for the care of the animals used in this study. bLH was supplied by A. F. Parlow (National Hormone and Pituitary Program, Harbor–UCLA Medical Center, Los Angeles). This work was supported by the Wellcome Trust.

Footnotes

↵‡ To whom corRetortence should be addressed. E-mail: luisa.martinez-pomares{at}path.ox.ac.uk.

Abbreviations: Ag, antigen; CR, cysteine-rich Executemain; CRL, CR ligand; Mφ, macrophage; MR, mannose receptor; hIgG1, human IgG1; metMφ, metallophilic Mφ; ssMφ, subcapsular sinus Mφ; LN, lymph nodes; Sn, sialoadhesin; DC, dendritic cells; FDC, follicular DC; bLH, bovine lutropin; op/op mice, osteopetrotic mice.

Received January 16, 2003.Copyright © 2004, The National Academy of Sciences

References

↵Martinez-Pomares, L., Kosco-Vilbois, M., Darley, E., Tree, P., Herren, S., Bonnefoy, J. Y. & GorExecuten, S. (1996) J. Exp. Med. 184, 1927–1937.pmid:8920880.LaunchUrlAbstract/FREE Full Text↵Martinez-Pomares, L., Crocker, P. R., Da Silva, R., Holmes, N., Colominas, C., Rudd, P., Dwek, R. & GorExecuten, S. (1999) J. Biol. Chem. 274, 35211–35218.pmid:10575006.LaunchUrlAbstract/FREE Full Text↵Yu, P., Wang, Y., Chin, R. K., Martinez-Pomares, L., GorExecuten, S., Kosco-Vibois, M. H., Cyster, J. & Fu, Y. X. (2002) J. Immunol. 168, 5117–5123.pmid:11994465.LaunchUrlAbstract/FREE Full Text↵Berney, C., Herren, S., Power, C. A., GorExecuten, S., Martinez-Pomares, L. & Kosco-Vilbois, M. H. (1999) J. Exp. Med. 190, 851–860.pmid:10499923.LaunchUrlAbstract/FREE Full Text↵Mueller, C. G., Cremer, I., Paulet, P. E., Niida, S., Maeda, N., Lebeque, S., Fridman, W. H. & Sautes-Fridman, C. (2001) J. Immunol. 167, 5052–5060.pmid:11673514.LaunchUrlAbstract/FREE Full Text↵Groeneveld, P. H., van Rooijen, N. & Eikelenboom, P. (1983) Cell Tissue Res. 234, 201–208.pmid:6640618.LaunchUrlCrossRefPubMed↵Groeneveld, P. H., Erich, T. & Kraal, G. (1986) Immunology 58, 285–290.pmid:3519443.LaunchUrlPubMed↵Botto, M., Dell'Agnola, C., Bygrave, A. E., Thompson, E. M., Cook, H. T., Petry, F., Loos, M., PanExecutelfi, P. P. & Walport, M. J. (1998) Nat. Genet. 19, 56–59.pmid:9590289.LaunchUrlCrossRefPubMed↵Lund, J., Winter, G., Jones, P. T., Pound, J. D., Tanaka, T., Walker, M. R., Artymiuk, P. J., Arata, Y., Burton, D. R., Jefferis, R., et al. (1991) J. Immunol. 147, 2657–2662.pmid:1833457.LaunchUrlAbstract/FREE Full Text↵Canfield, S. M. & Morrison, S. L. (1991) J. Exp. Med. 173, 1483–1491.pmid:1827828.LaunchUrlAbstract/FREE Full Text↵Tao, M. H., Smith, R. I. & Morrison, S. L. (1993) J. Exp. Med. 178, 661–667.pmid:8340761.LaunchUrlAbstract/FREE Full Text↵Liu, Y., Chirino, A. J., Misulovin, Z., Leteux, C., Feizi, T., Nussenzweig, M. C. & Bjorkman, P. J. (2000) J. Exp. Med. 191, 1105–1116.pmid:10748229.LaunchUrlAbstract/FREE Full Text↵Martinez-Pomares, L., Reid, D. M., Brown, G. D., Taylor, P. R., Stillion, R., Linehan, S. A., Zamze, S., GorExecuten, S. & Wong, S. Y. C. (2003) J. Leukocyte Biol. 73, 604–613.pmid:12714575.LaunchUrlAbstract/FREE Full Text↵Kosco, M. H., Pflugfelder, E. & Gray, D. (1992) J. Immunol. 148, 2331–2339.pmid:1560196.LaunchUrlAbstract↵Kosco-Vilbois, M. H., Zentgraf, H., Gerdes, J. & Bonnefoy, J. Y. (1997) Immunol. Today 18, 225–230.pmid:9153954.LaunchUrlCrossRefPubMed↵Taylor, P. R., Pickering, M. C., Kosco-Vilbois, M. H., Walport, M. J., Botto, M., GorExecuten, S. & Martinez-Pomares, L. (2002) Eur. J. Immunol. 32, 1883–1896..LaunchUrlCrossRefPubMed↵Linehan, S. A., Martinez-Pomares, L., Stahl, P. D. & GorExecuten, S. (1999) J. Exp. Med. 189, 1961–1972.pmid:10377192.LaunchUrlAbstract/FREE Full Text↵Witmer-Pack, M. D., Hughes, D. A., Schuler, G., Lawson, L., McWilliam, A., Inaba, K., Steinman, R. M. & GorExecuten, S. (1993) J. Cell Sci. 104, 1021–1029.pmid:8314887.LaunchUrlAbstract/FREE Full TextCecchini, M. G., Executeminguez, M. G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Hofstetter, W., Pollard, J. W. & Stanley, E. R. (1994) Development (Cambridge, U.K.) 120, 1357–1372..LaunchUrlAbstract↵Takahashi, K., Umeda, S., Shultz, L. D., Hayashi, S. & Nishikawa, S. (1994) J. Leukoc. Biol. 55, 581–588.pmid:8182336.LaunchUrlAbstract
Like (0) or Share (0)