Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells
Andrew R. Ferguson,
Michele E. Youd and
Ronald B. Corley
Department of Microbiology, Boston University School of Medicine, Boston, MA 02118, USA
Correspondence to: R. B. Corley; E-mail: rbcorley{at}bu.edu
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Abstract
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Secreted IgM and complement are important mediators in the optimal initiation of primary T-dependent humoral immune responses. Secreted IgM serves as a natural adjuvant by enhancing the immunogenicity of protein antigens, perhaps as a result of IgM's ability to facilitate antigen deposition onto follicular dendritic cells (FDCs) and promote rapid germinal center (GC) formation. To understand how IgM enhances adaptive immune responses, we investigated the mechanism by which IgM-containing immune complexes (IgM-IC) are transported to FDCs as a first step in GC formation. We demonstrate that IgM-IC localize first to the splenic marginal zone (MZ) where the IgM-IC bind MZ B cells in a complement and complement receptor (CR1/2) dependent process. MZ B cells then transport the IgM-IC into the follicle for deposition onto FDCs. Mice with reduced numbers of MZ B cells trap IgM-IC on FDC less efficiently, whereas mice with reduced numbers of follicular B cells trap IgM-IC normally. The functional elimination of MZ B cells abrogates the ability of FDCs to trap IgM-IC. Transfer of B cells with associated IgM-IC into naive mice results in deposition of IgM-IC onto FDC by MZ B cells. The results demonstrate an IgM and complement-dependent role for MZ B cells in the fate of antigen early in the initial phases of T-dependent immune responses. The data also establish an important role for CR1/2 on MZ B cells in the efficient binding and transport of IgM-IC to FDCs, which we suggest is an important first step in initiating adaptive immune responses.
Keywords: antigen transport, CD19, complement, complement receptors, germinal center
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Introduction
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Secreted IgM plays an important role in the initiation of adaptive humoral immune responses. Mice deficient in secreted IgM
have immune defects characterized by their inability to respond to low concentrations of protein antigens, delayed production of protective antibody responses to pathogens, and delayed germinal center formation (15). In the absence of secreted IgM, pathogens fail to concentrate into secondary lymphoid organs and instead disseminate into vital organs (6). The adjuvant activity of IgM is complement dependent and, consequently, mice deficient in components of the classical complement pathway or in the complement receptors CR1/2 (CD21/35) (7) have defects similar to those observed in
mice (814). Because specific IgM enhances immune responses to particulate and soluble antigens (5,1517), the defects in adaptive immune responses in
mice can be attributed, at least in part, to the failure of other secreted Ig isotypes to compensate for IgM function.
To investigate the role that IgM plays in enhancing immune responses, we established a system to evaluate the immunogenicity and fate of IgM-containing immune complexes (IgM-IC). Antigen associated with IgM, but not antigen alone, stimulated primary immune responses and primed for secondary immune responses (5). IgM-IC localized first in the splenic marginal zone (MZ) and then concentrated onto follicular dendritic cells (FDCs) within 16 h of i.v. injection. The localization of antigen on FDCs has been implicated in optimizing the formation of germinal centers (GC), memory B cell formation and somatic hypermutation (1821).
The complement receptors CR1/2 are important for the localization of antigen to FDCs (19,2225). Consistent with this, we found that FDC localization of IgM-IC was not observed in Cr2/ mice or in mice depleted of the complement component C3. Instead, IgM-IC localized exclusively to the MZ where they were bound by the phagocytic marginal zone macrophages (MZM) for removal (5). Interestingly, IgM-IC exhibited a different distribution on cells within the MZ in the presence of an intact complement system. In complement and CR1/2 sufficient mice, IgM-IC were not only associated with MZM, but with other cells in the MZ (5). Thus, both complement and CR1/2 play important roles in the fate of the IgM-IC in the MZ and in FDC localization. Collectively, these results suggested that the IgM-IC that were not associated with MZM in complement sufficient mice might instead be associated with a cell or cells responsible for the transport of IgM-IC from the MZ for deposition onto FDCs.
To test this hypothesis, we characterized the cells which bind IgM-IC, determined the mechanism by which IgM-IC are transported to FDCs and further examined the consequences of IgM on immune responses to antigen. We provide evidence that immunization with IgM-IC induces rapid GC formation. We also demonstrate that MZ B cells participate in the initiation of T-dependent immune responses as a result of their capacity to bind and transport IgM-IC into the B cell follicle, and deposit these IgM-IC onto FDCs. While their positioning in the MZ favors their initial interaction with IgM-IC, both in vivo and in vitro analyses demonstrate that MZ B cells are also particularly adept at binding large amounts of IgM-IC as a result of their increased expression of CR1/2. For binding and transport of IgM-IC, both complement and the CR1/2 are essential, providing a mechanistic basis for the role of complement and CR1/2 in the transport of IgM-IC from the MZ and deposition onto FDCs in the B cell follicle, initiating primary immune responses and facilitating GC formation.
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Methods
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Mice
C57BL/6J (B6), C57BL/6J-IghaThy1aGpi1a (B6-Igha) and CD19-deficient (CD19/) mice (26) were purchased from Jackson Laboratories (Bar Habor, ME). Breeding pairs of secreted IgM-deficient
mice (1) were provided by Dr Jianzhu Chen (Center for Cancer Research, MIT, Cambridge, MA). Breeding pairs of Cr2-deficient mice (12) were provided by Dr Ann Marshak-Rothstein (Department of Microbiology, Boston University School of Medicine) from mice originally provided by Dr Michael Carroll (The Center for Blood Research, Harvard Medical School, Boston, MA). IL-7/ mice (27) were obtained from DNAX Research Institute (Palo Alto, CA). B6-Igha, Cr2/, IL-7/ and
mice were bred and maintained in the Laboratory Animal Sciences Center, Boston University Medical Center. Mice of both sexes were used at 812 weeks of age. C3-depleted mice were produced by treatment with cobra venom factor (CVF) (Quidel Corp., San Diego, CA) as described (5). For in vivo depletion of MZ B cells, mice were injected i.v. with 30 ng pertussis toxin (PTx; List Biological Laboratories, Campbell, CA) 2 days before use as described (28).
Preparation and injection of IgM-IC in vivo
Mice were injected i.v. with a mixture of 1 µg each of NP-KLH (Biosearch Technologies, Novato, CA) and affinity purified pentameric B1-8 IgM mAbs or NP-KLH alone, exactly as previously described (5), to monitor GC formation. For immunohistochemistry (IH), 1520 µg NP7BSAbiotin (Biosearch Technologies) in a mixture with equivalent amounts of NP-specific IgM were injected i.v. This increased concentration was required for visualization of the antigen. While IgM-IC efficiently localize to FDC, similar amounts of antigen alone cannot be detected on FDCs (unpublished data) (5).
In vitro IgM-IC binding assay and transfer experiments
B cells were enriched from the spleens of B6 or B6-Igha mice by depletion of T cells with mixtures of anti-CD8 and anti-CD4 antibodies and complement (29). Macrophages were depleted by plastic adherence. IgM-IC were prepared by first incubating 1 µg each of NP7BSAbiotin and B1-8 pentameric IgM for 30 min at 37°C in DMEM containing 10% freshly isolated mouse serum as a source of complement, or with DMEM containing 10% heat inactivated (56°C, 30 min) mouse serum to deplete complement (30). 107 splenic B cells were then resuspended in preformed IgM-IC and incubated an additional 30 min at 37°C. Cells were then washed twice with PBS containing 1% FBS and resuspended for flow cytometry. For the transfer experiment, 5 x 107 cells were incubated with 2025 µg IgM-IC prepared as described above. After washing cells, the cells were resuspended in 250 µl PBS and injected i.v. into recipient mice as described in Results.
Immunohistochemistry
Staining of spleen sections was performed as previously described (5). Briefly, spleens were removed at the indicated times and snap frozen in O.C.T. compound (Tissue Tek, Torrance, CA) and stored at 80°C. Six-micrometer sections were cut (IEC Microtome, Needham, MA) and fixed in acetone at 20°C for 10 min. For staining, sections were incubated with 0.3% H2O2/PBS for 5 min then blocked with anti-Fc
II/III receptor antibody (2.4G2, BD Biosciences, San Diego, CA) diluted 1/100, or 3% normal rabbit or rat serum. All antibodies were diluted in 5% BSA in PBS. NP7BSAbiotin was visualized by alkaline phosphatase (AP)streptavidin (1/400; Molecular Probes, Eugene, OR) and developed with napthol ASMX phosphate/Fast blue BB base (Sigma, St Louis, MO). Visualization of antibody stains was accomplished using horseradish peroxidase (HRP)-conjugated secondary antibodies developed with 3-amino-9-ethylcarbazole (Sigma) and H2O2. Primary antibodies used included: anti-IgMaFITC (1/100), anti-B220FITC (1/100), anti-CD3FITC (1/100), anti-CD3biotin (1/100), anti-CD1dFITC (1B1; 1/50), anti-CD21/35FITC (1/50), anti-
(1/50) (all from BD Biosciences), MOMA-1 (1/25; Serotec, Raleigh NC), ERTR9 (1/200; BMA Biomedical, Augst, Switzerland), anti-FDCM1 (1/200; provided by Steve Bogan, Department of Pathology, Boston University School of Medicine) and anti-FDCM2 (1/200; Immunokontact, Frankfurt, Germany). GCs were identified using FITC-labeled peanut agglutinin (PNA; EY Laboratories, San Mateo, CA). Secondary antibodies included anti-FITCHRP (1/100; Roche Molecular Biochemicals, Indianapolis, IN), goat anti-rat IgGHRP (1/500; Jackson Immunoresearch, West Grove, PA) and goat anti-rat IgM (Fab')2HRP (1/1000; Jackson Immunoresearch). After staining, sections were covered with crystal mount (Biomedia, Foster City, CA) and visualized. Unless otherwise noted, a 10x objective was used for the initial magnification. In all figures, different antibody combinations were always performed on serial sections. For immunofluorescence, antibodies included anti-IgM-Texas Red (1/100; BD Biosciences) and anti-laminin (1/500; Sigma) visualized using an anti-rabbit IgG-FITC (1/100; Jackson Immunoresearch).
Flow cytometry
Splenocytes were isolated at the indicated times and stained as follows. The lymphocyte gate (as defined by forward and side scatter characteristics) was utilized for all analysis. Cells were analyzed using a FACScan (Becton Dickinson, Franklin Lakes, NJ) and FlowJo software (Tree Star, Ashland, OR). Antibodies utilized were anti-B220FITC (1/100), anti-B220biotin (1/100), anti-B220PE (1/100), anti-CD21/35FITC (1/100), anti-CD23PE (1/100) and anti-CD1dFITC or PE (1/50) (BD Biosciences). StreptavidinPE-Cy5 (1/200; BD Biosciences) was used to identify the NP antigen (NP7BSAbiotin) associated with lymphocytes, as well as to visualize biotin-labeled antibodies.
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Results
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IgM-IC promote rapid GC formation
Mice lacking secreted IgM fail to mount rapid antibody responses to antigens and exhibit delayed GC formation (1,2). Because the addition of IgM to T-dependent antigens restores the ability of these mice to mount primary and secondary immune responses (5), we first asked if these mice developed GCs. Mice
immunized with IgM-IC, but not antigen alone, developed GC as determined by PNA staining 7 days later (Fig. 1). These GC corresponded with intense regions of staining for the
light chain, as expected of the clonally restricted response to NP (31) and the pauciclonal nature of GC responses to this hapten (32). Therefore, one of the consequences of IgM-IC immunization is the rapid development of GC which contribute to the evolution of the immune response to T-dependent antigens.
IgM-IC associate with MZ B cells in vivo
In complement sufficient mice, IgM-IC are found first in the MZ and then localize to FDCs (5). To investigate how IgM-IC are transported from the MZ to FDCs, IgM-IC were injected into mice and the cell-associated IgM-IC in the spleen was analyzed 1 h later by flow cytometry. As shown in Fig. 2(A), MZ B cells, whether identified by their CD21/CD23 profile (CD21hiCD23lo) (33,34) or by CD1d expression (35,36), consistently bound more IgM-IC than the corresponding FO B cells. Neither immature B cells (CD21loCD23lo) nor T cells showed appreciable binding of IgM-IC (data not shown). These results indicate that MZ B cells efficiently bind blood-borne IgM-IC that filter through the spleen. As expected, IH showed that most of the IgM-IC were localized within the MZ 1 h after injection (Fig. 2B). However, some IgM-IC could already be detected inside the marginal sinus (lined by the MOMA-1+ marginal metallophilic macrophages) (37) and within the follicle in the first hour after injection (see arrow). Some of these IgM-IC were already associated with CD21hi cells, consistent with FDC localization.

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Fig. 2. IgM-IC transiently associate with MZ B cells in vivo. mice were injected with IgM-IC and spleens were collected 1 and 4 h later with half of the spleen frozen for IH and the other half processed for flow cytometry. (A) FACS analysis 1 h post-injection identifying MZ and FO B cells using a CD21/23 or B220/CD1d expression profile. IgM-IC were detected on these cells by streptavidinPE-Cy5 which recognizes NPBSAbiotin in the IgM-IC. The shaded area represents IgM-IC bound to B cells from injected mice while clear regions represent uninjected mice. (B) IH analysis of the other half of the spleen from (A) demonstrates the presence of IgM-IC (stained blue in this and all subsequent figures; see Methods) in the MZ, which lies outside of the MOMA-1+ marginal metallophilic macrophages (stained red-brown) surrounding the B cell follicle. The arrow indicates the presence of IgM-IC within the follicle. (C) Binding of IgM-IC to MZ B cells is not detected 4 h after injection, MZ and FO B cells were defined by the CD21/23 profile and analyzed for IgM-IC binding as described in (A). (D) By 4 h, most of the IgM-IC is concentrated around FDCs, as defined by the intense CD21/35 staining (red-brown). Experiments repeated more than five times gave similar results.
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To determine if IgM-IC remained associated with MZ B cells, spleens of mice were removed 4 h after injection and the amount of cell-associated IgM-IC was assessed. As shown in a representative experiment (Fig. 2C), we have consistently been unable to isolate B cells with associated IgM-IC 4 h after injection. At this time, much of the IgM-IC is found in the follicles associated with FDCs, as determined by IH (Fig. 2D). In other experiments, we have found that IgM-IC are associated with MZ B cells 2 h after injection, but at diminished levels compared with the 1 h time point as assessed by FACS (data not shown).
An intact complement system is required for efficient binding of IgM-IC to MZ B cells
IgM-IC do not localize to FDCs in complement-deficient and Cr2/ mice, but instead are slowly removed from the MZ by MZM (5). We reasoned, therefore, that a major determinant of the initial binding of IgM-IC to MZ B cells might be the interaction of complement components with CR1/2. To test this, C3-depleted or Cr2/ mice were injected with IgM-IC and, 1 h later, the association of IgM-IC with splenic B cells was assessed. As shown in Fig. 3, little if any IgM-IC bound to MZ or FO B cells (distinguished by their CD1d phenotypes) in either the C3-depleted (Fig. 3A) or Cr2/ (Fig. 3C) mice, indicating that C3 and CR1/2 play a crucial role in the binding of IgM-IC to these B cells. In agreement with previous results (5), all of the IgM-IC detected by IH was restricted to the MZ in these mice (Fig. 3B and D). Together, these results indicate that the binding of IgM-IC to MZ B cells is dependent on an intact complement system.

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Fig. 3. Complement and complement receptors are essential for the binding of IgM-IC to MZ B cells. (A and C) FACS analysis of splenocytes from a C3-depleted mouse (A) or Cr2/ mouse (C) 1 h after injection of IgM-IC. MZ and FO B cells were delineated using B220/CD1d staining and bound IgM-IC was visualized as described in Fig. 2(A). (B and D) IH analysis of the other half of the spleen used in (A) and (C) shows IgM-IC localized to the MZ, but without any IgM-IC inside the MOMA-1+ macrophage ring. This localization contrasts with the pattern in complement sufficient mice (see Fig. 2B).
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MZ B cells bind more IgM-IC than FO B cells in vitro
The results indicate that IgM-IC associate primarily, if not exclusively, with MZ B cells in vivo, and can be interpreted in at least two, non-mutually exclusive, ways. First, MZ and FO B cells might have similar capacities to bind IgM-IC, but the positioning of the MZ B cells within the MZ, the first site of encounter of blood borne IgM-IC, would favor IgM-IC binding to this B cell subset. Alternatively, MZ B cells might have an inherently greater capacity to bind IgM-IC than FO B cells due to their higher level of expression of CR2 (34). To discriminate between these alternatives, the ability of MZ and FO B cells to bind IgM-IC was compared in vitro. Since the presence of complement is essential for binding of IgM-IC to MZ B cells in vivo (Fig. 3A), IgM-IC were prepared in the presence of fresh mouse serum. As shown in Fig. 4(A), IgM-IC could bind both MZ and FO B cells. However, MZ B cells consistently bound more IgM-IC than FO B cells (estimated to be 510-fold more by MFI from >20 independent experiments), despite the fact that MZ B cells represent only a minor subset of splenic B cells. These results demonstrate that MZ B cells have a higher intrinsic capacity to bind IgM-IC.

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Fig. 4. MZ B cells bind more IgM-IC than FO B cells in vitro, and the binding is complement and CR1/2 dependent. (A) Analysis of the ability of MZ and FO B cells to bind IgM-IC in vitro. Splenic B cells were incubated with IgM-IC (1 µg each of NPBSAbiotin and anti-NP IgM) for 30 min at 37°C. The incubations were performed in the presence of fresh mouse serum as a source of complement (shaded area identified by +C), or with heat inactivated serum (clear area identified by C) as described in Methods. B cells incubated in media alone served as the negative control (represented by the dotted line). Flow cytometry was used to examine IgM-IC association with MZ and FO B cells, defined by their CD21/CD23 profile, as described in Fig. 2. (B) Comparison of the ability of B cells from normal (B6) and Cr2/ mice to bind IgM-IC. B cells were incubated with IgM-IC as described in (A). MZ and FO B cells were defined by CD1d gating on B220+ cells as described in Fig. 2.
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To confirm the dependence of IgM-IC binding on complement and CR1/2, the ability of B cells to bind IgM-IC was first compared when IgM-IC were prepared in the presence (fresh mouse serum) or absence (heat-inactivated serum) of complement. As shown in Fig. 4(A), the ability of both MZ and FO B cells to bind IgM-IC was severely impaired when IgM-IC were prepared in the absence of complement. In addition, the ability of both MZ and FO B cells from Cr2/ mice to bind IgM-IC was greatly diminished compared with mice expressing intact CR1/2. These results support the in vivo data demonstrating an important role for CR1/2 for the binding of IgM-IC by MZ B cells.
Role of MZ B cells for efficient trapping of IgM-IC on FDC
To examine the requirement for MZ B cells in the deposition of IgM-IC on FDC, several approaches were taken. First, we examined the fate of IgM-IC in the spleens of CD19/ mice, reported to be deficient in MZ B cells (36,38). CD19/ mice were injected with IgM-IC and spleens examined by IH 1 and 10 h later. As shown in Fig. 5(A), IgM-IC localized first to the MZ of these mice and, within 10 h, some IgM-IC were found on FDCs. However, the intensity of staining of IgM-IC in the splenic follicles of CD19/ mice was substantially reduced compared to B6 mice, suggesting that much less IgM-IC reached the FDCs of the CD19/ mice.


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Fig. 5. CD19/ mice have reduced numbers of MZ B cells and exhibit decreased efficiency in IgM-IC localization on FDC. (A) IH shows IgM-IC localization in CD19/ mice at 1 and 10 h after injection. At 1 h, IgM-IC localize to the MZ, but fewer IgM-IC localize to FDCs as compared with WT B6 mice. (B) FACS profile of B6 and CD19/ splenocytes showing association of IgM-IC in vivo to MZ B cells, as defined in Fig. 2(A). (C) In vitro binding of IgM-IC to B6 and CD19/ MZ (shaded) and FO (clear) B cells. (D) Immunofluorescence of uninjected B6 and CD19/ spleen sections with arrows identifying IgM+ cells in the MZ, demarcated by laminin staining. IgM appears red and laminin appears green.
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To confirm that CD19/ mice truly lacked MZ B cells, FACS analysis was performed (Fig. 5B). The number of MZ B cells was drastically reduced in these mice. However, some B cells did fall within the MZ gate, and these exhibited high levels of IgM-IC binding, consistent with that of MZ B cells from B6 mice (Fig. 5B, lower panels). This suggested that CD19/ mice have small numbers of functional MZ B cells. To substantiate their presence and function, an in vitro binding assay was performed. B cells from CD19/ mice that fell within the MZ B cell gate exhibited high levels of IgM-IC binding in vitro, at levels similar to that of MZ B cells from control mice (Fig. 5C). Finally, to verify that these putative MZ B cells could be found in the MZ of CD19/ mice, immunofluorescence was performed using laminin staining to demarcate the MZ (39). As shown in Fig. 5(D), spleen follicles of CD19/ mice contained IgM+ cells in the MZ, although in reduced numbers compared with normal mice. Therefore, although CD19/ mice have fewer MZ B cells, these can bind high levels of IgM-IC, and appear to be positioned normally around the marginal sinus. This conclusion is consistent with recent results indicating that MZ B cells are generated in CD19/ mice, but turnover rapidly (40). It is likely that these MZ B cells are involved in the deposition of IgM-IC on FDCs in these mice (Fig. 5A).
As an alternative approach, we took advantage of the ability of pertussis toxin (PTx) to functionally deplete MZ B cells (28). Mice were treated with PTx and 2 days later the fate of IgM-IC was examined. The depletion of MZ B cells by PTx was confirmed by reductions in CD21hiCD23lo B cells (Fig. 6A). Small numbers of splenocytes from these mice were found in the MZ gate, and these bound detectable amounts of IgM-IC, as compared with uninjected controls (Fig. 6A, lower panels). We then examined the fate of IgM-IC in the spleens of these mice. Although IgM-IC localized to FDC 18 h after injection in control mice, IgM-IC were completely absent in the spleens of PTx-treated mice (Fig. 6B). FDCs were found in these mice, indicating that their presence was not compromised by PTx treatment. In addition, significant amounts of IgM-IC were localized to the MZ 1 h after injection (Fig. 6B, lower panels), indicating that treatment of mice with PTx did not alter the ability of IgM-IC to localize to the MZ of the splenic follicles. However, the IgM-IC were never observed within the marginal sinus, as determined by MOMA-1 staining. This indicates that, in PTx-treated mice, IgM-IC are trapped normally within the MZ, but do not subsequently migrate into the follicles.
To determine if the presence of MZ B cells is sufficient for FDC trapping, IgM-IC were injected into IL-7-deficient mice, which contain a high frequency of MZ B cells and significantly reduced numbers of FO B cells (41) (Fig. 6C). IgM-IC localized to FDCs in the spleens of these mice, indicating that IgM-IC are deposited onto FDC even when FO B cells constitute only a small percentage of the splenic B cells.
MZ B cells transfer IgM-IC to FDC
While the previous results indicate that IgM-IC are bound by MZ B cells, and that the presence of MZ B cells is required for the deposition of IgM-IC onto FDCs, they do not prove that MZ B cells are actively transporting the IgM-IC later found on the FDC. To examine the ability of MZ B cells to transport IgM-IC, we initially tried to isolate these cells by cell sorting and use them to transfer IgM-IC to recipient mice in vivo. However, we have found that the transport and deposition of IgM-IC is inhibited during the sorting process (unpublished observations). As an alternative approach, we used unsorted B cells in an adoptive transfer experiment, and then sought to distinguish the phenotype of the cells that were transferring IgM-IC to FDCs. To distinguish transferred B cells from endogenous B cells, Igh congenic strains of mice were used.
B6-Igha B cells were isolated and incubated with IgM-IC in the presence of fresh or heat inactivated serum. After IgM-IC binding, B cells were washed to remove unbound IgM-IC, and transferred i.v. into a recipient B6 mouse. Spleens were removed 12 h later and processed for IH. Spleen sections were first examined for the presence of IgM-IC within the B cell follicles. As expected, no IgM-IC were found within follicles of mice injected with B cells incubated with IgM-IC formed in the absence of complement (Fig. 7A). However, IgM-IC were found within the B cell follicles in mice that had received B cells incubated with IgM-IC in the presence of complement. To determine if the transferred B cells homed to the B cell follicle and associated with FDCs, spleen sections from these mice were stained for IgM-IC and for the expression of IgMa, which detects only the transferred B cells. Representative sections are shown in Fig. 7(B). Transferred B cells were not only found within the B cell follicle, but intimately associated with structures having dendritic morphology with associated IgM-IC, consistent with FDCs. Interestingly, the IgM-IC on the transferred B cells was frequently capped on one pole, directed toward the dendrites (Fig. 7B, closed arrow), suggesting that the B cells might be transferring the IC to the FDCs. These data conclusively demonstrate that B cells alone can serve as the source of IgM-IC found subsequently on FDC.

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Fig. 7. MZ B cells bind and transfer IgM-IC to FDC. (A) B cells were isolated from Igha B6 mice and incubated with IgM-IC as explained in Fig. 4(A). IgM-IC laden B cells (4 x 107) were transferred i.v. into B6 mice. Spleens were harvested 12 h later and prepared for IH. Stains were performed as described. (B) Transferred B cells deposit IgM-IC onto FDCs. The utilization of Igha B cells enabled tracking of these cells using an anti-IgMaFITC antibody. Notice the cluster of IgMa+ cells around areas of IgM-IC deposition. Higher magnification (100x) shows FDC dendrites loaded with IgM-IC (identified by dashed arrow) as well as IgMa+ cells transferring IgM-IC to an empty FDC dendrite (identified by closed arrow). (C) Stains for CD1d and FDC-M2 to visualize transferred MZ B cells depositing IgM-IC onto FDCs. CD1d+ cells encompass the B cell follicle and are found inside the B cell follicle. FDC-M2 stain confirms the presence of IgM-IC on FDC. At higher magnification (100x), MZ B cells (CD1d+) can be observed depositing IgM-IC onto FDC processes (arrows).
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Serial sections from the spleens of mice injected with the transferred cells carrying IgM-IC were next stained with CD1d, to distinguish MZ from FO B cells, and the FDC marker FDC-M2. CD1d staining revealed a characteristic ring around the follicle, as expected for the positioning representative of MZ B cells (Fig. 7C). Since few transferred cells (as defined by IgMa staining) were found in the MZ, these CD1d+ cells must represent the endogenous MZ B cells. Higher magnifications of these sections showed the presence of CD1d+ cells inside the follicle clustered around FDC, as illustrated in the lower panels. Some of the CD1d+ positive cells contained IgM-IC and therefore can clearly be identified as the transferred cells. These cells were juxtaposed with FDCs, as shown by the FDC-M2 staining in the serial section (lower panel, right). Together, these results are consistent with a role for MZ B cells in the transport and deposition of IgM-IC onto FDCs.
While the transfer experiments demonstrate the ability of MZ B cells to transport IgM-IC to FDCs, they do not rule out the possible contribution of FO B cells. To address this issue, B cells from PTx-treated mice, which are deficient in MZ B cells (Fig. 6), were used as a source of donor B cells. As shown in Fig. 8(A), significant amounts of IgM-IC were found on FDCs when normal but not PTx-treated mice were used as a source of B cells. Nevertheless, small amounts of IgM-IC were detectable in some of the follicles of recipients of PTx-treated cells. These IgM-IC could have been transferred (albeit inefficiently) by FO B cells, or they could have been transported to FDCs after transfer to recipient MZ B cells. To distinguish between these two possibilities, the transfer experiment was repeated using PTx-treated mice as recipients. As shown in Fig. 8(B), only B cells from untreated mice were capable of transferring IgM-IC to recipient FDCs. We conclude that MZ B cells are uniquely capable of transporting and depositing IgM-IC to FDCs.

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Fig 8. MZ B cells are uniquely capable of transferring IgM-IC to FDCs. B cells were isolated from untreated or PTx-treated mice, incubated with IgM-IC and complement as described in Fig. 4(A) and transferred into untreated (A) or PTx-treated (B) recipient B6 mice. Spleens were removed 12 h (A) or 8 h (B) later and processed for IH. In (B), the boxed sections are shown at higher magnification in the right panels. Arrows identify regions in which IgM-IC are localized to FDCs.
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Discussion
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One of the hallmarks of T-dependent antibody responses is the development of GC which promote affinity maturation and memory B cell development (20,21). IgM has a potent adjuvant effect by promoting primary immune responses to low doses of antigen that do not normally elicit such responses, and priming for memory B cell formation (5). The IgM-induced response is also characterized by the induction of GC (Fig. 1). One of the earliest events that can be detected in mice immunized with IgM-IC, but not antigen alone, is the deposition of the IgM-IC onto FDCs (5) (Fig. 2), suggesting that this might reflect one of the initial steps in the immunopotentiating effect of IgM.
The trapping of antigen on FDCs has long been thought to play an important role in immune responses (19,4244). The role of antigen deposition on FDC for GC function has recently been called into question (45) based on studies that show that B cells from mice that lack secreted antibodies (46) or lymphotoxin
, which lack FDCs (47), still undergo somatic hypermutation. However, large doses of antigen with adjuvant were used in these studies, and it has been reported that somatic mutations do not arise in mice lacking FDCs in the absence of adjuvants (47,48). These data argue that FDCs are most important in orchestrating adaptive antibody responses under conditions in which antigen is limiting, such as those used in the current study. Given that responses only initiate when antigen is complexed with IgM, we suggest that the deposition of IgM-IC onto FDCs not only promotes GC formation and function (Fig. 1), but jump starts the immune response as well.
Previous studies have established the requirements for secreted IgM (16) and an intact complement system (814) in the optimal initiation of adaptive T-dependent immune responses, but the exact mechanism(s) by which these mediators fulfill these functions has remained largely unknown. Our observations link the requirement for IgM and complement in a process in which IgM-IC activate the complement cascade, promoting the binding of these complexes to CR1/2 on MZ B cells. Several lines of evidence indicate that these MZ B cells are then responsible for transporting the complexes to the FDC. Reductions in MZ B cells either significantly (CD19/) or entirely (PTx-treated mice) prevent IgM-IC deposition onto FDCs. Transfer studies prove that B cells can serve as the sole source of IgM-IC, indicating that they alone bind sufficient IgM-IC for FDC localization (Fig. 7). Furthermore, these transfer experiments demonstrate that the donor B cells must contain functional MZ B cells to enable the efficient transfer of IgM-IC to FDCs (Fig. 8). This supports the idea that only MZ B cells are competent to transport IgM-IC to FDCs. Notably, in the transfer experiments, MZ B cells with capped IgM-IC could be found in close juxtaposition with FDC dendrites, supporting a proposed role for MZ B cells in the localization of IgM-IC on FDCs. Interestingly, at times beyond 2 h after i.v. injection, we are unable to recover MZ B cells, or any cells for that matter, with associated IgM-IC (Fig. 2B), even though these cells are clearly present by IH (see Fig. 7). One possible explanation is that these MZ B cells are intertwined within the FDC network and, like FDCs themselves (49), cannot be released into cell suspension without procedures that incorporate enzymatic digestion. Together, these data provide strong evidence for a major role for MZ B cells in the deposition of IgM-IC onto FDCs.
MZ B cells have been established as a major component of the rapid antibody response to both T-independent blood borne pathogens and T-dependent antigens (28,33,5052). MZ B cells are positioned to interact with these antigens by virtue of their localization around the marginal sinus of the splenic follicle. Our results suggest that MZ B cells not only function in the early phases of the antibody responses to antigens, but also play an important role in the initiation of the T-dependent immune response by virtue of their ability to bind, transport and deposit IgM-IC onto FDCs. Their ability to carry out this function is not only facilitated by their unique localization at the marginal sinus, but by the high levels of CR1/2 present on these cells, which enables them to efficiently interact with blood borne IgM-IC.
CR1/2 appears to be the major receptor on MZ B cells involved in the initial binding of IgM-IC. In the absence of CR1/2, little binding of IgM-IC can be detected over background, suggesting that other potential receptors, such as the Fc
/µ receptor (53) or the B cell receptor (BCR), play limited roles, at least in initiating IgM-IC binding. The deposition of IgM-IC onto FDCs in the absence of the BCR co-receptor, CD19 (54,55), suggests a role for CR1/2 in IgM-IC transport independent of signaling through CD19. Interestingly, the Cr2/ mice used in the current study have a truncated form of the CR2 receptor on the cell surface (56). From our data it is clear that this truncated receptor binds little if any IgM-IC and is not functional. Therefore, we conclude that CR1/2 is the major receptor involved in the binding of the IgM-IC to MZ (and FO) B cells. Our data also suggest that MZ B cells have been able to exploit the elevated levels of membrane CR1/2 to bind larger amounts of IgM-IC, increasing their efficiency in transporting these IC into the B cell follicle for deposition onto FDC.
The idea that specific cells transport IC into splenic follicles is not new. A number of earlier studies examined this issue using aggregated IgG or large IgG-IC, and gave rise to competing theories on the mechanism by which IC reach FDCs. These included passive diffusion (57,58) and active transport by macrophages (59), migrating FDC precursors (60,61), or lymphocytes (6267). The interactions of the IgG with IgG Fc receptors (68) on various cells types may have complicated the ability to interpret results from these studies. Moreover, in many of these studies, trafficking of the IC was studied in immunized recipients, in contrast with our own studies. Indeed, we have found that IgG antibodies cannot substitute for IgM when using naive mice, suggesting that IgM has a unique, non-redundant role in antigen deposition in non-immune mice (A.R.Ferguson and R.B.Corley, manuscript in preparation). This is consistent with an IgM-specific defect in mice devoid of secreted IgM, but expressing other secreted isotypes (14).
Two mechanisms have been proposed to explain the positioning of MZ B cells within the MZ. Cyster and colleagues (69) demonstrated the retention of MZ B cells through integrin-mediated adhesion. Ravetch and colleagues (70) found that MZM expressing MARCO are responsible for the retention of MZ B cells. The BCR-dependent interaction of MZ B cells with antigens has been shown to result in migration of these cells from the MZ to the TB interface, followed by the subsequent relocation of these cells to the red pulp as differentiated plasmablasts [reviewed in (71,72)]. In the current study, we found that the interaction of MZ B cells with IgM-IC causes their migration from the MZ into the follicle, with subsequent deposition of IgM-IC onto FDCs. The means by which IgM-IC causes the release of MZ B cells from retention and directs trafficking into the follicle is unknown. In this regard, the results using PTx-treated mice are of interest. PTx inhibits G protein signaling involved in chemotaxis (73). While the low residual numbers of MZ B cells in CD19/ mice enable IgM-IC deposition onto FDCs, this is not the case in PTx-treated mice. This suggests that chemotatic signals might be involved in directing MZ B cells into the follicles, once they are released from retention signals. A delineation of the signals involved in the release and migration of MZ B cells following IgM-IC interactions will not only be important in determining their fate, but will provide insights into how the deposition of IgM-IC onto FDCs plays a role in stimulating the onset of the adaptive humoral immune response.
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Acknowledgements
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The authors wish to thank Jianzhu Chen (Center for Cancer Research, MIT, Cambridge, MA), Michael Carroll (The Center for Blood Research, Harvard Medical School, Boston, MA), DNAX Research Institute (Palo Alto, CA) and Ann Marshak-Rothstein (Boston University School of Medicine) for providing mouse strains used in this study, and our colleagues Steve Bogen, Lee Wetzler and Ann Marshak-Rothstein for some of the reagents used in these studies. This work was supported by the National Institutes of Health grant AI31209 and by the Aid for Cancer Research, Boston, MA. A.R.F. and M.E.Y. were recipients of an Institutional Immunology Training grant award from the NIAID (T32-AI07309).
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Notes
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Transmitting editor: D. Fearon
Received 24 March 2004,
accepted 12 July 2004.
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References
|
---|
- Boes, M., Esau, C., Fischer, M. B., Schmidt, T., Carroll, M. and Chen, J. 1998. Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM. J. Immunol. 160:4776.[Abstract/Free Full Text]
- Ehrenstein, M. R., O'Keefe, T. L., Davies, S. L. and Neuberger, M. S. 1998. Targeted gene disruption reveals a role for natural secretory IgM in the maturation of the primary immune response. Proc. Natl Acad. Sci. USA 95:10089.[Abstract/Free Full Text]
- Boes, M., Prodeus, A. P., Schmidt, T., Carroll, M. C. and Chen, J. 1998. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188:2381.[Abstract/Free Full Text]
- Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. E., Herzenberg, L. A. and Chen, J. 2000. B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. J. Exp. Med. 192:271.[Abstract/Free Full Text]
- Youd, M. E., Ferguson, A. R. and Corley, R. B. 2002. Synergistic roles of IgM and complement in antigen trapping and follicular localization. Eur. J. Immunol. 32:2328.[CrossRef][ISI][Medline]
- Ochsenbein, A. F., Fehr, T., Lutz, C., Suter, M., Brombacher, F., Hengartner, H. and Zinkernagel, R. M. 1999. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286:2156.[Abstract/Free Full Text]
- Ahearn, J. M. and Fearon, D. T. 1989. Structure and function of the complement receptors, CR1 (CD35) and CR2 (CD21). Adv. Immunol. 46:183.[ISI][Medline]
- Pepys, M. B. 1974. Role of complement in induction of antibody production in vivo. Effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J. Exp. Med. 140:126.[Abstract/Free Full Text]
- Heyman, B., Wiersma, E. J. and Kinoshita, T. 1990. In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody. J. Exp. Med. 172:665.[Abstract]
- Fischer, M. B., Ma, M., Goerg, S. et al. 1996. Regulation of the B cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157:549.[Abstract]
- Wessels, M. R., Butko, P., Ma, M., Warren, H. B., Lage, A. and Carroll, M. C. 1995. Studies of group B streptococcal infection in mice deficient in complement C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc. Natl Acad. Sci. USA 92:11490.[Abstract]
- Ahearn, J. M., Fischer, M. B. et al. 1996. Disruption of the Cr2 locus results in a reduction in B1a cells and in an impaired B cell response to T-dependent antigen. Immunity 4:251.[ISI][Medline]
- Molina, H., Holers, V. M., Li, B., Fang, Y.-F., Mariathasan, S., Goellner, J., Strauss-Schoenberger, J., Karr, R. W. and Chaplin, D. D. 1997. Markedly impaired humoral response in mice deficient in complement receptors 1 and 2. Proc. Natl Acad. Sci. USA 93:3357.[ISI]
- Cutler, A. J., Botto, M., van Essen, D., Rivi, R., Davies, K. A., Gray, D. and Walport, M. J. 1998. T cell-dependent immune response in C1q-deficient mice: defective interferon gamma production by antigen-specific T cells. J. Exp. Med. 187:1789.[Abstract/Free Full Text]
- Henry, C. and Jerne, N. K. 1968. Competition of 19S and 7S antigen receptors in the regulation of the primary immune response. J. Exp. Med. 128:133.[Abstract/Free Full Text]
- Heyman, B., Andrighetto, G. and Wigzell, H. 1982. Antigen-dependent IgM-mediated enhancement of the sheep erythrocyte response in mice. Evidence for induction of B cells with specificities other than that of the injected antibodies. J. Exp. Med. 155:994.[Abstract/Free Full Text]
- Heyman, B., Pilström, L. and Shulman, M. J. 1988. Complement activation is required for IgM-mediated enhancement of the antibody response. J. Exp. Med. 167:1999.[Abstract]
- Tew, J. G., Phipps, R. P. and Mandel, T. E. 1980. The maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells. Immunol. Rev. 53:175.[ISI][Medline]
- Liu, Y. J., Grouard, G., de Bouteiller, O. and Banchereau, J. 1996. Follicular dendritic cells and germinal centers. Int. Rev. Cytol. 166:139.[ISI][Medline]
- Tarlinton, D. 1998. Germinal centers: form and function. Curr. Opin. Immunol. 10:245.[CrossRef][ISI][Medline]
- McHeyzer-Williams, L. J., Driver, D. J. and McHeyzer-Williams, M. G. 2001. Germinal center reaction. Curr. Opin. Hematol. 8:52.[CrossRef][ISI][Medline]
- Yoshida, K., van den Berg, T. K. and Dijkstra, C. D. 1993. Two functionally different follicular dendritic cells in secondary lymphoid follicles of mouse spleen, as revealed by CR1/2 and FcR gamma II-mediated immune-complex trapping. Immunology 80:34.[ISI][Medline]
- Van den Berg, T. K., Yoshida, K. and Dijkstra, C. D. 1995. Mechanism of immune complex trapping by follicular dendritic cells. Curr. Top. Microbiol. Immunol. 201:49.[ISI][Medline]
- Qin, D., Wu, J., Carroll, M. C., Burton, G. F., Szakal, A. K. and Tew, J. G. 1998. Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J. Immunol. 161:4549.[Abstract/Free Full Text]
- Fang, Y., Xu, C., Fu, Y.-X., Holers, V. M. and Molina, H. 1998. Expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific IgG response. J. Immunol. 160:5273.[Abstract/Free Full Text]
- Rickert, R. C., Roes, J. and Rajewsky, K. 1997. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25:1317.[Abstract/Free Full Text]
- von Freeden-Jeffry, U., Vieira, P., Lucian, L. A., McNeil, T., Burdach, S. E. and Murray, R. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract]
- Guinamard, R., Okigaki, M., Schlessinger, J. and Ravetch, J. V. 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat. Immunol. 1:31.[CrossRef][ISI][Medline]
- Rifkin, I. R., Leadbetter, E. A., Beaudette, B. C., Kiani, C., Monestier, M., Shlomchik, M. J. and Marshak-Rothstein, A. 2000. Immune complexes present in the sera of autoimmune mice activate rheumatoid factor B cells. J. Immunol. 165:1626.[Abstract/Free Full Text]
- Polley, M. J. and Muller-Eberhard, H. J. 1966. Chemistry and mechanism of action of complement. Prog. Hematol. 5:2.[Medline]
- Reth, M., Imanishi-Kari, T. and Rajewsky, K. 1979. Analysis of the repertoire of anti-(4-hydroxy-3-nitro-phenyl)acetyl (NP) antibodies in C57BL/6 mice by cell fusion II. Characterization of idiotopes by monoclonal anti-idiotope antibodies. Eur. J. Immunol. 9:1004.[ISI][Medline]
- Jacob, J., Przylepa, J., Miller, C. and Kelsoe, G. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178:1293.[Abstract]
- Oliver, A. M., Martin, F., Gartland, G. L., Carter, R. H. and Kearney, J. F. 1997. Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses. Eur. J. Immunol. 27:2366.[ISI][Medline]
- Takahashi, K., Kozono, Y., Waldschmidt, T. J., Berthiaume, D., Quigg, R. J., Baron, A. and Holers, V. M. 1997. Mouse complement receptors type 1 (CR1;CD35) and type 2 (CR2;CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice. J. Immunol. 159:1557.[Abstract]
- Amano, M., Baumgarth, N., Dick, M. D., Brossay, L., Kronenberg, M., Herzenberg, L. A. and Strober, S. 1998. CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms. J. Immunol. 161:1710.[Abstract/Free Full Text]
- Makowska, A., Faizunnessa, N. N., Anderson, P., Midtvedt, T. and Cardell, S. 1999. CD1high B cells: a population of mixed origin. Eur. J. Immunol. 29:3285.[CrossRef][ISI][Medline]
- Kraal, G. and Janse, M. 1986. Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody. Immunology 58:665.[ISI][Medline]
- Martin, F. and Kearney, J. F. 2000. Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19 and btk. Immunity 12:39.[ISI][Medline]
- Wang, S. L., Kutsche, M., DiSciullo, G., Schachner, M. and Bogen, S. A. 2000. Selective malformation of the splenic white pulp border in L1-deficient mice. J. Immunol. 165:2465.[Abstract/Free Full Text]
- Otero, D. C., Anzelon, A. N. and Rickert, R. C. 2003. CD19 function in early and late B cell development: I. Maintenance of follicular and marginal zone B cells requires CD19-dependent survival signals. J. Immunol. 170:73.[Abstract/Free Full Text]
- Carvalho, T. L., Mota-Santos, T., Cumano, A., Demengeot, J. and Vieira, P. 2001. Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7(/) mice. J. Exp. Med. 194:1141.[Abstract/Free Full Text]
- Mandel, T. E., Phipps, R. P., Abbot, A. and Tew, J. G. 1980. The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53:29.[ISI][Medline]
- Klaus, G. G., Humphrey, J. H., Kunkl, A. and Dongworth, D. W. 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53:3.[ISI][Medline]
- Kosco-Vilbois, M. H. 2003. Are follicular dendritic cells really good for nothing? Nat. Rev. Immunol. 3:764.[CrossRef][ISI][Medline]
- Haberman, A. M. and Shlomchik, M. J. 2003. Reassessing the function of immune-complex retention by follicular dendritic cells. Nat. Rev. Immunol. 3:757.[ISI][Medline]
- Hannum, L. G., Haberman, A. M., Anderson, S. M. and Shlomchik, M. J. 2000. Germinal center initiation, variable gene region hypermutation and mutant B cell selection without detectable immune complexes on follicular dendritic cells. J. Exp. Med. 192:931.[Abstract/Free Full Text]
- Matsumoto, M., Lo, S. F., Carruthers, C. J. et al. 1996. Affinity maturation without germinal centres in lymphotoxin-alpha-deficient mice. Nature 382:462.[CrossRef][ISI][Medline]
- Fu, Y. X., Molina, H., Matsumoto, M., Huang, G., Min, J. and Chaplin, D. D. 1997. Lymphotoxin-alpha (LTalpha) supports development of splenic follicular structure that is required for IgG responses. J. Exp. Med. 185:2111.[Abstract/Free Full Text]
- Schnizlein, C. T., Kosco, M. H., Szakal, A. K. and Tew, J. G. 1985. Follicular dendritic cells in suspension: identification, enrichment and initial characterization indicating immune complex trapping and lack of adherence and phagocytic activity. J. Immunol. 134:1360.[Abstract/Free Full Text]
- Song, H. and Cerny, J. 2003. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. J. Exp. Med. 198:1923.[Abstract/Free Full Text]
- Oliver, A. M., Martin, F. and Kearney, J. F. 1999. IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells. J. Immunol. 162:7198.[Abstract/Free Full Text]
- Martin, F., Oliver, A. M. and Kearney, J. F. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14:617.[CrossRef][ISI][Medline]
- Shibuya, A., Sakamoto, N., Shimizu, Y. et al. 2000. Fc
/µ receptor mediates endocytosis of IgM-coated microbes. Nature Immunol. 1:441.[CrossRef][ISI][Medline]
- Fearon, D. T. and Carter, R. H. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13:127.[CrossRef][ISI][Medline]
- Tedder, T. F., Inaoki, M. and Sato, S. 1997. The CD19CD21 complex regulates signal transduction thresholds governing humoral immunity and autoimmunity. Immunity 6:107.[ISI][Medline]
- Hasegawa, M., Fujimoto, M., Poe, J. C., Steeber, D. A. and Tedder, T. F. 2001. CD19 can regulate B lymphocyte signal transduction independent of complement activation. J. Immunol. 167:3190.[Abstract/Free Full Text]
- Groeneveld, P. H., Eikelenboom, P. and van Rooijen, N. 1983. Mechanism of follicular trapping: similarities and differences in trapping of antibody-complexed antigens and carbon particles in the follicles of the spleen. J. Reticuloendothel. Soc. 33:109.[ISI][Medline]
- Laman, J. D., Kors, N., Van Rooijen, N. and Claassen, E. 1990. Mechanism of follicular trapping: localization of immune complexes and cell remnants after elimination and repopulation of different spleen cell populations. Immunology 71:57.[ISI][Medline]
- White, R. G., French, V. I. and Stark, J. M. 1970. A study of the localisation of a protein antigen in the chicken spleen and its relation to the formation of germinal centres. J. Med. Microbiol. 3:65.[ISI][Medline]
- Szakal, A. K., Holmes, K. L. and Tew, J. G. 1983. Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131:1714.[Abstract/Free Full Text]
- del Cacho, E., Gallego, M., Arnal, C. and Bascuas, J. A. 1995. Localization of splenic cells with antigen-transporting capability in the chicken. Anat. Rec. 241:105.[ISI][Medline]
- Brown, J. C., De Jesus, D. G., Holborow, E. J. and Harris, G. 1970. Lymphocyte-mediated transport of aggregated human gamma-globulin into germinal centre areas of normal mouse spleen. Nature 228:367.[ISI][Medline]
- Oldfield, S., Lortan, J. E., Hyatt, M. A. and MacLennan, I. C. 1988. Marginal zone B cells and the localisation of antigen on follicular dendritic cells. Adv. Exp. Med. Biol. 237:99.[Medline]
- Veerman, A. J. and van Rooijen, N. 1975. Lymphocyte capping and lymphocyte migration as associated events in the in vivo antigen trapping process. An electron-microscopic autoradiographic study in the spleen of mice. Cell Tissue Res. 161:211.[ISI][Medline]
- Gray, D., Kumararatne, D. S., Lortan, J., Khan, M. and MacLennan, I. C. 1984. Relation of intra-splenic migration of marginal zone B cells to antigen localization on follicular dendritic cells. Immunology 52:659.[ISI][Medline]
- Heinen, E., Braun, M., Coulie, P. G. et al. 1986. Transfer of immune complexes from lymphocytes to follicular dendritic cells. Eur. J. Immunol. 16:167.[ISI][Medline]
- Braun, M., Heinen, E., Cormann, N., Kinet-Denoel, C. and Simar, L. J. 1987. Influence of immunoglobulin isotypes and lymphoid cell phenotype on the transfer of immune complexes to follicular dendritic cells. Cell. Immunol. 107:99.[ISI][Medline]
- Ravetch, J. V. and Bolland, S. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19:275.[CrossRef][ISI][Medline]
- Lu, T. T. and Cyster, J. G. 2002. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297:409.[Abstract/Free Full Text]
- Karlsson, M. C., Guinamard, R., Bolland, S., Sankala, M., Steinman, R. M. and Ravetch, J. V. 2003. Macrophages control the retention and trafficking of B lymphocytes in the splenic marginal zone. J. Exp. Med. 198:333.[Abstract/Free Full Text]
- Martin, F. and Kearney, J. F. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2:323.[ISI][Medline]
- MacLennan, I. C., Toellner, K. M., Cunningham, A. F., Serre, K., Sze, D. M., Zuniga, E., Cook, M. C. and Vinuesa, C. G. 2003. Extrafollicular antibody responses. Immunol. Rev. 194:8.[CrossRef][ISI][Medline]
- Cyster, J. G. and Goodnow, C. C. 1995. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J. Exp. Med. 182:581.[Abstract]