The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Y.
Right arrow Articles by Tryggvason, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Y.
Right arrow Articles by Tryggvason, K.
The Journal of Immunology, 2005, 175: 8173-8180.
Copyright © 2005 by The American Association of Immunologists

Defective Microarchitecture of the Spleen Marginal Zone and Impaired Response to a Thymus-Independent Type 2 Antigen in Mice Lacking Scavenger Receptors MARCO and SR-A1

Yunying Chen*, Timo Pikkarainen*, Outi Elomaa*, Raija Soininen{dagger}, Tatsuhiko Kodama{ddagger}, Georg Kraal§ and Karl Tryggvason2,*

* Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden; {dagger} Department of Medical Biochemistry, Biocenter Oulu, University of Oulu, Oulu, Finland; {ddagger} Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and § Department of Cell Biology, Free University, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by the marginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritoneal macrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of the scavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organization of the splenic marginal zone, and causes a significant reduction in the size of the resident peritoneal macrophage population, possibly due to changes in adhesion and migration capacity. In mice lacking both MARCO and SR-A these effects are even more apparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appearance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a clear delay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the various subsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challenged with a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockout mice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being important scavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction with endogenous ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The marginal zone (MZ)3 of the spleen has a central role for the initiation of both innate and adaptive immunity against blood-borne pathogens. Directly surrounding the white pulp, the lymphoid compartment of the spleen, the MZ contains cells that are involved in both thymic-independent and -dependent immune responses (1). Among them are dendritic cells that can process Ags in the MZ, leading to activation and expression of appropriate chemokine receptors, so that the dendritic cells will enter the white pulp and can activate recirculating T cells to ensure efficient local immune responses (2, 3, 4). In addition, the MZ harbors a population of activated B cells, the MZ B cells, that preferably locate here and are programmed to initiate a fast and intense Ab response to blood-borne viral and bacterial agents, making them key players in the early response to pathogens in the bloodstream (5, 6).

The most predominant cell population of the MZ is a special type of macrophage, characterized by specific expression of the pattern recognition receptors macrophage receptor with a collagenous structure (MARCO) and specific intracellular adhesion molecule-grabbing nonintegrin receptor 1 (SIGNR1), which have been shown to bind a broad range of microbial Ags (7, 8, 9, 10). This fact, together with their strategic position in the bloodstream of the MZ makes these macrophages important for efficient removal of pathogens, but it may also lead to a concentration of pathogenic material which can promote activation of macrophages and the MZ B cells, thus combining innate and adaptive immune responses. The marginal zone macrophage (MZM) is distinct from the marginal metallophilic macrophage (MMM) located at the inner face of the MZ sinus, bordering the MZ and the follicular areas of the white pulp. These cells uniquely express sialoadhesin (sialic acid-binding Ig-like lectin-1 (Siglec-1)), which functions as a receptor for microbial polysaccharides, in addition to playing a role in adhesive interactions with other leukocytes (11, 12, 13).

It has recently become clear that both B cells and the MZ macrophages are indispensable for the integrity and proper function of the MZ. In the absence of B cells during ontogeny, both the MMMs and the MZMs, as well as mucosal addressin cellular adhesion molecule-1 (MAdCAM-1)-positive sinus-lining cells in the MZ sinus are absent (14). But also the integrity and function of an established MZ was dependent on the presence of B cells, as shown in models where B cells could be depleted by removal of the BCR subunit Ig{alpha} and in a transgenic model in which all B cells were gradually depleted due to overexpression of the TNF family member CD70 (15). In contrast, the MZMs are essential for retention and trafficking of the MZ B cells, as shown by selective disruption of the inositol phosphatase SHIP (16). Interestingly, it was demonstrated in the same study that the scavenger receptor MARCO showed activity for endogenous ligands on the MZ B cells and was associated with the retention of the B cells in the MZ (16).

MARCO has a highly restricted expression pattern in adult mice living under pathogen-free conditions (7). Besides the MZMs, it is constitutively expressed in macrophages in the medullary cord of lymph nodes and in peritoneal macrophages. However, following bacterial infection, MARCO is readily induced in other macrophage populations such as in the spleen and liver (10). In contrast, the related scavenger receptor SR-A is normally widely expressed in macrophages, and it can be found in macrophages in the MZ and the red pulp. Scavenger receptor A (SR-A) null mice have been found to be more susceptible than control mice to infection with Listeria monocytogenes and Staphylococccus aureus, indicating a role for SR-A in antimicrobial host-defense mechanisms (17, 18).

In this study, we have analyzed the role of MARCO and SR-A in the formation and function of the spleen using gene-targeted mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

The generation of MARCO–/– mice will be described elsewhere. These mice, as well as SRA–/– mice (18), were backcrossed to the C57BL/6 (B6) strain for >10 generations. To generate mice deficient for both MARCO and SR-A (double-KO), homozygous MARCO–/– mice were mated with homozygous SR-A–/– mice. The resulting double heterozygotes (F1) were then intercrossed to generate F2 mice. Double-KO F2 generation mice were then mated together to produce mice used for the experiments described in this study. To propagate the double-KO colony, the mice were backcrossed to the B6 strain. As controls, wild-type mice from breedings of the MARCO+/– mice were used. All mutant mice appeared normal and fertile in a pathogen-free environment. All mice studies were approved by the institutional review committee.

Tissue stainings and immunofluorescence microscopy

Fresh OCT-embedded tissues were frozen in liquid nitrogen and stored at –70°C. Cryosections of 8 µm were fixed in acetone for 10 min. After incubation in 10% normal serum from the species in which the secondary Ab was generated, the sections were incubated with the primary Ab, followed by several washes in PBS and incubation with a fluorescently labeled secondary Ab. For double staining, tissue sections were first stained for one of the Ags, then incubated in 20% normal rat serum again, and subsequently stained for the other Ag by first incubating with a biotinylated mAb and then with fluorescently labeled streptavidin.

The following rat-anti-mouse mAbs were used: ED31, an anti-MARCO Ab (19); ERTR9, which recognizes the C-type lectin SIGNR1 expressed in the MZ (20); MOMA-1, recognizing a recently identified Siglec-1 Ag in the MMMs (Ref.21 ; G. Kraal, unpublished data); MECA367, an anti-MAdCAM-1 mAb staining the endothelial cells lining the MZ sinus; F4/80 (clone CI:A3-1; Serotec), pan-macrophage marker; and an anti-IgD Ab (clone 11-26; Southern Biotechnology Associates). Additionally, biotinylated rabbit anti-mouse IgM (µ-chain specific; Zymed Laboratories) was used.

Binding of the unconjugated primary Abs was detected with Alexa Fluor 488- or 546-conjugated goat anti-rat Abs (Molecular Probes). Biotinylated primary Abs were detected with Alexa Fluor 594-conjugated streptavidin (Molecular Probes) or with FITC-conjugated streptavidin (DakoCytomation).

Isolation of cells from the peritoneal cavity and cell counting

Resident peritoneal cells were isolated by rinsing the cavity with DMEM containing 10% FCS or with PBS. Thioglycolate-elicited macrophages were isolated in the same manner 4 days after i.p. injection of 1 ml of 3% Brewer’s thioglycolate. Macrophages were isolated from other cell types by exploiting their ability to strongly adhere to glass or plastic. Cells were plated in DMEM/FCS for 2 h on glass coverslips, after which unattached and loosely adherent cells were removed by washing extensively with PBS. The spreading of the macrophages was observed under phase-contrast microscopy. Cells were fixed at indicated time points in 4% paraformaldehyde, and stained with the Abs as described above. Actin cytoskeleton was visualized by staining with rhodamine-conjugated phalloidin (Molecular Probes). Cell nuclei were stained by 4',6'-diamidino-2-phenylindole (DAPI) nucleic acid stain (Molecular Probes).

For macrophage counting, cells collected from the peritoneal cavity were cytospun (~1 x 105 cells) onto normal microscope slides (5 min, 600 rpm). After drying, cells were fixed in acetone and stained with DAPI and the F4/80 mAb. DAPI was used to visualize all leukocytes, while F4/80 only visualized macrophages. Cell numbers were counted under a fluorescence microscope with a x20 objective. A minimum of 20 randomly selected fields was counted for each sample. The number of F4/80-positive cells divided by DAPI-positive cells represented the proportion of macrophages in the leukocyte population.

In some assays, equal volumes of peritoneal lavage fluid were plated on 24-well culture plates for 2 h, and adherent cells were quantitated, after first removing nonadherent cells by several washes with PBS. For quantitation, the cells were fixed with glutaraldehyde, stained with crystal violet, rinsed with water, and solubilized in 2% SDS. The absorbances were measured at 595 nm.

Migration assay

Migration activity of resident peritoneal macrophages was assayed using the Transwell two-chamber system (Costar, 8-µm pore size, 6.5-mm insert diameter). Resident peritoneal cells were harvested with PBS, washed once with DMEM containing 0.2% BSA and 15 mM HEPES (pH 7.4), and resuspended at 0.5 x 106 cells/ml in the same medium. One hundred microliters of a cell suspension was applied into the upper chamber. The lower chamber contained 10% FCS in DMEM (600 µl). After incubation for 5 h at 37°C, cells on the upper side of the membrane were removed with a cotton tip and three rinses with PBS. Cells on the underside of the membrane were fixed with methanol overnight at 4°C, and stained with F4/80 and DAPI. The membranes were mounted on glass slides, and cells were counted under a fluorescence microscope with a x20 objective.

To evaluate the number of peritoneal macrophages in the cell suspension applied into the upper chamber, an aliquot of the cell suspension was plated for 5 h on a glass coverslip, after which the coverslip was rinsed twice with PBS, and the attached cells were fixed in methanol and stained with F4/80 and DAPI.

Macrophage depletion from the spleen

Liposome-entrapped dichloromethylene diphosphonate (clodronate) suspension was obtained from Clodronateliposomes (Free University). To deplete cells from the spleen, 0.2-ml aliquots of the suspension were injected i.v. into each mouse (22). Reappearance of macrophages and the other MZ cell populations was monitored 4, 8, 11, 16, 21, 35, and 67 days after the treatment, by staining frozen sections with the various Abs described above. At least two mice per genotype were examined at each time point. This experiment was repeated twice.

Ab responses to pneumococcal polysaccharides

Pneumo23, a 23-valent pneumococcal vaccine containing 25 µg of the capsular polysaccharides from Streptococcus pneumoniae serotypes 1–5, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F in 0.5 ml NaCl with 0.25% phenol, was obtained from Aventis Pasteur. A 500-µl sample of the vaccine diluted 1/25 in 0.9% NaCl was injected i.p. to 5–9 mice per genotype in each experiment. The experiment was performed twice. Blood samples were taken by puncturing the tail artery at days 0, 7, 14, and 63 after the immunization from the same mice over time. Sera were stored at –20°C until analyzed. Serum anti-Pneumo23 IgM and IgG3 Igs were measured by ELISA. ELISA plates were coated with 100 µl of the vaccine at the concentration of 2.3 µg/ml PBS for 2 h at 37°C. After washing four times with PBS/0.05% Tween 20, the wells were incubated in 1% BSA to block the remaining binding sites. Then, 100 µl of the serum dilution (diluted in PBS/0.05% Tween 20) containing 0.5% BSA) was applied, and the anti-vaccine Abs were allowed to bind for 2 h at 37°C or overnight at 4°C. When testing anti-vaccine IgM Igs, we used a serum dilution 1/1000. In case of anti-vaccine IgG3, a serum dilution of 1/10 was used. These were found to be the proper dilutions in pilot experiments. Each serum sample was tested in duplicate. All bleedings were tested simultaneously. After the incubations, the wells were washed several times with PBS/Tween 20 and incubated with biotinylated rabbit anti-mouse IgM (Zymed Laboratories) or biotinylated monoclonal rat anti-mouse IgG3 (BD Pharmingen) for 90 min at 37°C. After washings with PBST, the wells were incubated in HRP-conjugated streptavidin (Pierce; diluted 1/10,000 in PBST) at room temperature for 20 min, and then washed again. A substrate solution sample of 100 µl, a 1:1 mixture of reagent A (H2O2) and reagent B (tetramethylbenzidine) (R&D Systems), was added into each well, and the color was allowed to develop for 10–20 min. The reaction was terminated by adding 50 µl of 2 N H2SO4. Absorbance values were read in a microplate reader at 450 nm and corrected by values obtained at 570 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effects of loss of MARCO and SR-A on spleen marginal zone macrophages

We were interested in finding out whether the loss of MARCO, or both MARCO and SR-A, affects the microarchitecture of the spleen MZ. The anti-SIGNR1 mAb ERTR9 was used to visualize the MZMs, since these cells, with only minor exceptions, display both MARCO and SIGNR1 in normal adult wild-type mice (7, 19). Staining of the different genotypes for SIGNR1 revealed that the lack of MARCO, or both MARCO and SR-A, indeed resulted in defects in the MZ microarchitecture (Fig. 1). The SIGNR1-expressing cells that, in wild-type mice, form a distinct zone of three to four cell layers of macrophages between the red and white pulp compartments showed a more diffuse staining pattern in the MARCO-KO and the double-KO mice. Fewer numbers of SIGNR1-positive cells were found in the KO mice than in their wild-type controls, and the cells were not tightly adhered to each other or the MZ sinus, resulting in a more discontinuous MZ and a "gap" between the MZ macrophage layer and the sinus. This phenotype was even more severe in the double-KO mouse, indicating that both MARCO and SR-A are needed for the intactness of the MZ macrophage layer. Indeed, in mice lacking only SR-A, the number of SIGNR1-expressing cells was clearly reduced but not to the extent as found in the MARCO-KO and the double-KO mice, and the cells also showed more adherence to each other and the marginal sinus. That the reduction in numbers of the SIGNR1-positive cells in the MZ of the KO mice was due to a reduction in actual cell numbers and not to that of SIGNR1 expression was verified by staining of spleen sections for acid phosphatase, a method identifying macrophages based on their enzyme activity, irrespective of receptor expression (1). As seen in Fig. 2, the reduction in numbers of the MZMs was particularly evident in the case of the double-KO mice.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 1. Genetic ablation of MARCO (MARCO-KO), SR-A (SR-A-KO) or both of these scavenger receptors (double-KO), alters the number and microarchitecture of the MZMs in adult spleen. The MZMs (green) were identified with a mAb recognizing SIGNR1, a lectin-like transmembrane protein. The endothelial cell layer lining the marginal sinus (red) was stained with a mAb against MAdCAM-1. As compared with wild-type mice, there are fewer numbers of MZMs in the single-KO mice and even fewer MZMs in mice deficient for both receptors. Note also the more sparse distribution of the MZMs in the KO mice.

 


View larger version (81K):
[in this window]
[in a new window]
  		FIGURE 2. Decreased number of MZMs in the double-KO mice as determined by staining for acid phosphatase activity. Spleen cryosections from wild-type and the double-KO mice were stained for this intracellular enzyme, a pan-macrophage marker that is also expressed in the MZMs at significant levels. As can be observed, the number of positive cells in the MZ is clearly lower in the double-KO than in wild-type mice.

 
In contrast to the effects on the MZMs, deletion of MARCO and SR-A affected neither the pattern of the MMMs on the inner side of the marginal sinus nor the presence of the MZ B cells, identified, respectively, by Siglec-1 expression and as cells expressing high levels of IgM and being negative for IgD (data not shown).

Absence of MARCO and SR-A affects spleen marginal zone organization during ontogeny

Because the absence of MARCO and SR-A affects the number and organization of the MZMs in adult spleen, we wished to examine whether their absence also influences the MZ development during ontogeny. At the day of birth (0 postpartum (pp)), when the MZ was still absent, MARCO-expressing cells were found dispersed throughout the spleen in wild-type mice (Fig. 3A). These cells were not positive for the pan-macrophage marker F4/80 data (not shown), indicating that the MZ and the red pulp macrophages of adult mice are two different cell populations already during the spleen development. The MARCO-positive MZ started to appear at day 3, having a more clear pattern at day 7 (Fig. 3A).



View larger version (108K):
[in this window]
[in a new window]
  		FIGURE 3. Neonatal development of the spleen MZ. A, Staining of spleen sections from wild-type mice indicates that MARCO- (red) and MAdCAM-1-expressing cells (green) are dispersed throughout the spleen at the day of birth (0 pp). At day 3 pp, both cell populations are already concentrating in the interface of the red and white pulp. A clear MZ pattern is established during the next few days. B, Formation of the MAdCAM-1-positive marginal sinus in the KO mice lags behind that in wild-type mice. The results indicate that the formation of the sinus rim is delayed by ~3 days in the MARCO-KO mice and by ~6 days in the double-KO mice. C, Staining of neonatal day 12 spleen sections for the MZ macrophage markers SIGNR1 and Siglec-1. This staining reveals that the appearance of the SIGNR1-expressing MZMs and the Siglec-1-expressing MMMs is markedly delayed in the KO mice. After day 12 pp the number of SIGNR1- and Siglec-1-positive cells increases in all three genotypes, but the number of SINGR1-positive cells in the KO mice never reaches that in wild-type mice (as shown in Fig. 1).

 
As was the case for MARCO, the MAdCAM-1-expressing cells were found dispersed throughout the spleen at the day of birth. The formation of the MAdCAM-1-positive marginal sinus also coincided with that of the MARCO-positive MZ in wild-type mice (Fig. 3A). Supporting the idea that there exists a relationship in the development of these two structures, we found that the formation of the MAdCAM-1-positive sinus was delayed in the KO mice. Thus, whereas in wild-type neonates the MAdCAM-1-positive sinus was clearly visible in a circular pattern from day 3 onward, MAdCAM-1 staining showed a circular pattern in the MARCO-KO mice first at about day 7, and in the double-KO mice at postnatal day 9 (Fig. 3B).

Expression of SIGNR1 and Siglec-1 started much later than that of MARCO or MAdCAM-1 during the neonatal spleen development, and they were not detected before day 7, the day when the MARCO- and MAdCAM-1-expressing cells already formed a clear MZ pattern. However, a more continuous rim-like MZ staining with these two markers was not seen before day 12 in wild-type mice. In the absence of MARCO, or both MARCO and SR-A, there was a significant delay in the appearance of the SIGNR1- and Siglec-1-positive MZ macrophage populations. Thus, as seen in Fig. 3C, at postnatal day 12, when a typical MZ pattern for SIGNR1 and Siglec-1 was found in wild-type mice, the expression of SIGNR1 and Siglec-1 was just starting in the MARCO-KO mice. Even less signal was detected in the double-KO mice. In case of each genotype, the staining pattern detected at about day 20 corresponded to that seen in adult mice (Fig. 1).

Delayed maturation of the MZ after liposome-induced cell depletion in the KO mice

Considering the apparent differences in the appearance of the various macrophage populations during the spleen development when MARCO and SR-A are not expressed, we wished to explore whether the rehabitation of splenic macrophages from bone marrow precursor monocytes is also affected. For that purpose, we injected clodronate liposomes i.v. into adult wild-type and KO mice, which leads to a rapid depletion (within 24 h) of all spleen red pulp and MZ macrophages, while macrophages in the white pulp stay intact (22). Such depletion is normally followed by a differential renewal of the splenic macrophages from blood monocytes, so that the red pulp macrophages reappear within 1 wk and the MZ cells later. The transient absence of the macrophages also leads to the temporary disappearance of the MZ B cells and to the loss of MAdCAM-1 expression.

Following liposome administration, completeness of the macrophage depletion was confirmed by staining spleen sections for F4/80 (data not shown). Examination of wild-type mice 8 days after the liposome-treatment revealed that F4/80- and MARCO-positive cells had started to reappear in the red pulp. The number of the MARCO-positive cells increased in the red pulp during the following days, and the first of them started to localize to the MZ around 16 days after the depletion. At around day 21 the typical ring-like MZ MARCO-staining became obvious, and there were only some MARCO-positive cells left in the red pulp. At day 35 the MARCO-staining pattern was indistinguishable from that seen in untreated mice (Fig. 4A).



View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 4. Reappearance of the different MZ cell populations after cell depletion by a single i.v. injection of clodronate-containing liposomes. A, The characteristic concentric MZ distribution pattern of MARCO can be detected ~15–20 days after the liposome treatment. When the spleen sections are stained with the mAb ED31 recognizing the scavenger receptor cysteine-rich domain of MARCO 4 days after the liposome treatment, only some fibrous staining left from depleted cells can be detected (data not shown). Similar fibrous staining can be detected at day 8. Furthermore, some MARCO-positive cells are found in the red pulp at this time point. The number of the MARCO-positive cells in the red pulp increases significantly at day 11. By day 16, significant numbers of the MARCO-positive cells have already migrated to the MZ, and a MARCO-positive MZ can be distinguished. At day 21, the characteristic ring-like MZ distribution pattern of MARCO is very obvious. At day 35, ED31 gives a very similar staining pattern to that seen in an untreated mouse. B, The different MZ cell populations appear in the KO mice with delayed kinetics as compared with similarly treated wild-type mice. The situation at day 35 after the liposome treatment is shown as an example. The panels represent the following stainings: upper row, the MZM marker SIGNR1; middle row, the MMM marker Siglec-1; lower row, the MZ B cells (IgMhigh/IgDlow; IgM, green; IgD, red). It is not shown here, but very similar staining patterns with their untreated control mice can be seen at the last time point examined, day 67.

 
As was the case with the MZ development during ontogeny, formation of the MARCO-positive MZ preceded the expression of SIGNR1 and Siglec-1 during the recovery process after the liposome-treatment. Furthermore, similarly to the situation in ontogeny, SIGNR1 and Siglec-1 appeared during this recovery process only in the MZ. Siglec-1 appeared somewhat earlier than SIGNR1 and was detected already at day 16 after the liposome-treatment. SIGNR1 expression first appeared at day 21 (data not shown).

Compared with wild-type mice, liposome depletion in the KO mice led to a significant delay in the reappearance of the MZ macrophage populations. The expression of MAdCAM-1 was also severely affected. As exemplified in Fig. 4B, at day 35 after the treatment, when the typical MZ MARCO-staining pattern is detected (Fig. 4A), staining for SIGNR1 and Siglec-1 revealed a clear MZ staining pattern in wild-type mice, but not in the MARCO-KO and the double-KO mice (Fig. 4B). In these two KO strains, SIGNR1 and Siglec-1 expression was only starting to appear. Surprisingly, in contrast to the situation in ontogeny where no effects of MARCO were seen on the MZ B cells, the reappearance of these cells after the liposome-treatment was severely affected in the KO mice. The MZ B cells were found to have started reappearing 16 days after the depletion in wild-type mice but not in the KO mice. The situation at day 35 is shown in Fig. 4B. At the latest time point tested (67 days after the depletion), the replenishment of all MZ cell populations had reached in all three genotypes similar levels as in their untreated counterparts (data not shown).

Finally, it is worth noting here that when LPS was injected into depleted wild-type mice three days after the liposome-treatment, a strong MARCO expression was observed on white pulp macrophages (data not shown). This does not occur when adult mice are challenged by LPS, or if LPS was administered 1 wk after the liposome-treatment. In these cases, a strong up-regulation of MARCO in the red pulp macrophages was seen, indicating that LPS is able to reach the white pulp when the MZ and red pulp macrophages are eliminated. These observations suggest that MARCO expression is probably induced in vivo in all macrophage subpopulations encountering a pathogenic molecule.

The absence of MARCO leads to cellular changes

The defects found in the architecture of the MZ in the MARCO-KO and the double-KO mice could be attributed to defects in adhesion or spreading of the macrophages in the absence of MARCO and SR-A proteins. In studies with peritoneal macrophages from SR-A KO mice, defects in their spreading properties have been reported (18). Because ectopic expression of MARCO can induce formation of long dendritic processes in different cell lines (23), we examined the spreading properties of macrophages from the MARCO-KO and the double-KO mice. When plated on glass coverslips in the presence of serum for 24 h, spreading of the resident peritoneal macrophages from the MARCO- and double-KO mice was significantly impaired compared with the corresponding wild-type cells (Fig. 5, C–F), which constitutively express MARCO (Fig. 5A) (and SR-A). We also consistently observed that smaller numbers of resident peritoneal macrophages could be recovered from the MARCO-KO and double-KO mice than from wild-type mice (Fig. 5G). To see whether MARCO plays a role in the recruitment of new macrophages to the peritoneum, influx was induced using the thioglycollate-induced peritonitis model. However, the newly recruited macrophages did not express MARCO (Fig. 5B). This might be because the recruited cells are not mature macrophages. Notably, neither was LPS (alone or together with IFN-{gamma}) able to induce MARCO-expression in this cell population in vivo or in vitro (data not shown), although it rapidly induces MARCO expression in many resident macrophage subpopulations (10, 19, 24).



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 5. Effects of MARCO and SR-A ablation on peritoneal macrophages. Resident (A) or thioglycollate-elicited (B) macrophages collected from the peritoneal cavity of wild-type mice were cultured on glass coverslips for 24 h and stained with the mAb ED31 for MARCO (red). All cells were visualized by nuclear DAPI staining (blue). Resident peritoneal macrophages express MARCO. Only 10% of the peritoneal macrophages collected 4 days after the thioglycollate-injection were found to express MARCO. Because the thioglycollate-treatment increases the size of the peritoneal macrophage population by ~10-fold, this suggests that the elicited cells do not express MARCO. C–F, Resident peritoneal macrophages from the different mouse strains were grown on coverslips for 24 h in the complete culture medium (C, wild-type; D, MARCO-KO; E, SR-A-KO; F, double-KO). After fixation with paraformaldehyde, cells were visualized by staining for F-actin (red). These panels clearly demonstrate the defective spreading properties of the KO cells. G, The percentage of F4/80-positive cells in the cytospun total peritoneal leukocyte population. The results indicate that the KO mice exhibit a marked reduction in the number of the resident peritoneal macrophages (p < 0.001 by Student’s t test).

 
We then examined the migratory ability of the resident peritoneal macrophages from the double-KO and wild-type mice in a two-chamber migration system in which the cells were allowed to migrate for 5 h from serum-free medium toward medium containing 10% FCS. Both cell populations exhibited low chemotactic responsiveness, but in each of the several experiments we found higher numbers—2- to 3-fold or more—of the double-KO macrophages than their wild-type counterparts on the lower side of the membrane. These findings support the possibility that lack of MARCO and SR-A affects the retention of macrophages in the peritoneal cavity.

A role for MARCO in thymus-independent type 2 (TI-2) Ag responses

The spleen plays a major role in protection against infections with encapsulated bacteria, such as S. pneumoniae (25, 26, 27). Protection is mainly provided by Abs against capsular polysaccharides (PSs) (28). Capsular PSs belong to the class of T-independent type 2 Ags against which the MZ with its macrophages and B cells plays an important role (1). We therefore wished to compare the Ab responses against a preparation of capsular PSs from 23 commonly occurring serotypes of pneumococcal bacteria in wild-type and the MARCO-KO and the double-KO mice. Because TI-2 Ags elicit primarily production of IgM and IgG3 in mice (29, 30, 31), only the levels of these two Ab classes were measured. Serum IgM and IgG3 levels were measured 1, 2, and 9 wk after injection of a single dose of capsular PSs. The results for IgM are shown in Fig. 6A, and represent at each time point the absorbance values of pooled sera at one dilution. For each genotype, sera from 10–15 mice immunized in two sets of injections were pooled. The results indicate higher anti-PS IgM response in wild-type mice than in the MARCO- or the double-KO mice. The double-KO mice showed a very low response, but these mice had higher anti-PS IgM levels before immunization than the other genotypes. The levels of total IgM Abs were also highest in this group (data not shown). In the case of IgG3, there was a large degree of mouse-to-mouse variability ranging from no response to high response even within each genotype. This was true particularly for the KO mice, in fact, when based on the median value virtually no response was seen in mice deficient in both MARCO and SR-A (Fig. 6B).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 6. Impaired responses against a pneumococcal polysaccharide vaccine in the KO mice. Wild-type and the different KO mice were immunized i.p. with 25 µg of a 23-valent pneumococcal vaccine, Pneumowax, and serum anti-Pneumo23 IgM and IgG3 Ab levels were measured at the indicated times. The results are shown in absorbances at one serum dilution. In the case of IgM (A), they represent at each time point the absorbance values of pooled sera collected from mice immunized in two independent experiments (n: wild type, 15; MARCO-KO, 13; SR-A-KO, 10; double-KO, 12). In the case of IgG3 (B), the median absorbance values from one of two similar experiment are shown. Each experimental group consisted of five to nine mice ({triangleup}, wt; {square}, MARCO-KO; *, SR-A-KO; {circ}, double-KO).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MARCO and SR-A, two important scavenger receptors with a broad ligand specificity, play an important role in the recognition of pathogens and the initiation of the innate immune activation. Interestingly, interaction with self-ligands may be involved in the localization and migration of cells expressing the receptors, as suggested from the effects of SR-A on adhesive and spreading properties of macrophages, and from the fact that ectopic expression of MARCO led to dramatic cell shape changes in transfected cells.

We therefore focused in this study on the effects of ablation of MARCO and both MARCO and SR-A on the integrity of the spleen, especially since the MZMs uniquely express MARCO, but also significant levels of SR-A (32).

Two types of defects in the MZ of the KO mice were found: their MZMs were much more scattered than the ones in wild-type mice, and their number was reduced. These structural defects in the MZ were even more severe in the double MARCO/SR-A-KO mice than in the MARCO-KO mice, indicating that both MARCO and SR-A are needed for the proper structural integrity of this area.

The ontogeny studies and the effects of liposome depletion revealed an interesting spatiotemporal relationship between MARCO and SIGNR1 expression. First, MARCO was found to be expressed very early, much earlier than SIGNR1, in the spleens of wild-type mice both during ontogeny and after the liposome-treatment, first appearing on the red pulp macrophages. Second, a significant delay in the appearance of SIGNR1 expression was found in the KO mice compared with similarly treated wild-type mice. Strikingly, SIGNR1 expression was not detected in the red pulp macrophages, but only in the MZMs. This suggests that the expression of MARCO (and possibly SR-A) is necessary for the proper development and organization of the MZ and precedes further differentiation of the MZMs as evidenced by the expression of SIGNR1.

Data to support such a sequential development come from studies with the osteopetrotic mutant mice, which are defective in the production of M-CSF. In these mice, the SIGNR1-positive MZMs and the Siglec-1-positive MMMs are absent, whereas the MARCO-expressing MZMs are present, indicating that additional growth requirements are needed for further differentiation of the MARCO-expressing macrophages (24, 33).

A possible mechanism for the structural defects in the MZ of the spleen could lie in the fact that MARCO is necessary for the proper adhesion and retention of the cells within the MZ through interaction with local structural components. This notion is supported by our findings that peritoneal macrophages from the MARCO-KO mice show defects in adhesion and spreading, and possibly also in cell retention, because the KO mice were found to have significantly reduced numbers of resident peritoneal macrophages. One possibility is that if the cells have defects in their adhesion and spreading capabilities, their lifespan could be affected. In contrast, the results from the migration assays point to the possibility that the efflux rate of macrophages out of the peritoneum is higher in the KO mice. Further studies will be needed to investigate these possibilities.

Our findings on the effects of MARCO and SR-A are in line with other reports showing that a complex interplay exists between the various cell types found in the MZ, including the different macrophage subpopulations, B cells and even the stromal sinus-lining cells. In particular, it appears that genetic defects leading to absence or low numbers of the MZMs coincide with defects in the MAdCAM-1-positive sinus-lining cells. For example, ablation of such genes as those encoding TNF, p55TNF-R, RelB, and Bcl-3 results in phenotypes where the numbers of the SIGNR1-positive MZMs are reduced drastically, and MAdCAM-1 is either completely missing or expressed at very low levels (33, 34, 35, 36). Similarly, mice lacking the transcription factor NKX2.3, which is required for MAdCAM-1 expression, were found to have reduced numbers of Siglec-1-positive MMMs, as well as drastically reduced numbers of SIGNR1-positive macrophages (37). Furthermore, these latter cells were not restricted any more to the MZ but were scattered in the red pulp.

In addition, there clearly is an interplay between B cells and macrophages, which is necessary for the proper organization of the MZ, as well as for the retention and function of both of these cell populations. The absence of signaling through lymphotoxin {beta} on B cells led to a reduction in size of the MZ B cell-population, but also to that of the SIGNR1-, Siglec-1-, and MAdCAM-1-positive cell populations (38). Related to this finding, a recent study with several KO models revealed that B cells are not only important for the development but also for the maintenance of the MZ macrophage subpopulations (15). Another recent study has shown that the interaction between MARCO and a cell surface determinant on the MZ B cells contributes to the retention of the MZ B cells within the MZ (16). This is in contrast to our ontogeny studies where the appearance of the MZ B cells showed no difference in the absence of MARCO but is in line with our liposome depletion studies, which also argue for a role of MARCO in the retention of the MZ B cells. It may well be the case that the importance of this interaction for the MZ B cell retention is not uncovered in the chronic loss-of-gene function situation, but only in an acute situation, such as the recovery process after the liposome treatment.

We observed that the ablation of MARCO led to an impaired response against a TI-2-Ag. After i.v. injection, these types of Ags are rapidly captured in the spleen by the MZMs and the MZ B cells via, respectively, SIGNR1- and complement-mediated processes (39, 40, 41). A previous study with Pyk-2-deficient mice demonstrated an important role for the MZ B cells in the Ab responses to one type of TI-2-Ag, Ficoll (41), but the role of the MZMs in this regard is controversial. In one study elimination of the MZM cells with clodronate-containing liposomes resulted in a strong decrease of the Ab response to TNP-Ficoll (42), but this could not be confirmed in other studies, including those where the MZMs were eliminated with the toxin gelonin coupled to the anti-SIGNR1 Ab ERTR9 (39, 43). In contrast, it is known that there is an age-associated decline in Ab responses to the pneumococcal PS in humans as well as in mice, and one reason for this phenomenon, according to in vitro studies, is a spleen accessory cell deficiency (44). The accessory cells can be depleted by adherence on plastic or by passage through a Sephadex column, indicating a macrophage-like nature of these cells. The exact role of the accessory cells in the vaccine response is not known, but they may have a role in the generation of a proper microenvironment for the MZ B cells, because the response in the aged mice spleen cells could be restored by supplementation with a variety of cytokines (44). It is of interest to note in this regard that staining of spleen section from aged mice for MARCO or SIGNR1 indicates significantly reduced numbers of MZMs compared with young adult mice (data not shown).

In addition to the MZ B cells, the peritoneal B1 B cells, the major produces of natural Abs, also participate in TI-2-responses (4, 45). In particular, when mice are challenged i.p. with a low dose of a TI-2-Ag, these cells are the first and probably the only exposed and responding B cell population (45). Notably, primed peritoneal macrophages have been found to effectively support B1 cell differentiation (4). We did not see any differences in the size of B1 cell population between the genotypes (data not shown). However, it cannot be excluded that the reduction in the number of peritoneal macrophages is one reason to the lowered TI-2 response in the KO mice.

In summary, our data show that in addition to their role as classical pattern recognition receptors involved in the uptake of pathogens, MARCO and, to a lesser extent, SR-A, play a role in the structural organization and cellular interactions of the marginal zone of the spleen, most likely through interactions with self ligands, and that such interactions are crucial for the functioning of the spleen and an optimal homeostasis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the Swedish Research Council and the Foundation for Strategic Research. Back

2 Address correspondence and reprint requests to Dr. Karl Tryggvason, Department of Medical Biochemistry and Biophysics, Division of Matrix Biologi, Karolinska Institutet, Scheeles Väg 2, B1, plan 4, S-17177 Stockholm, Sweden. E-mail address: karl.tryggvason{at}mbb.ki.se Back

3 Abbreviations used in this paper: MZ, marginal zone; DAPI, 4',6'-diamidino-2-phenylindole; KO, knockout; MAdCAM-1, mucosal addressin cellular adhesion molecule-1; MARCO, macrophage receptor with a collagenous structure; M-CSF, monocyte-colony stimulating factor; MMM, marginal metallophilic macrophage; MZ, marginal zone, MZM, marginal zone macrophage; pp, postpartum; PS, polysaccharide; Siglec-1, sialic acid-binding Ig-like lectin-1; SIGNR1, specific intracellular adhesion molecule-grabbing nonintegrin receptor-1; SR-A, scavenger receptor A; TI-2, thymus-independent type 2. Back

Received for publication April 13, 2005. Accepted for publication October 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kraal, G.. 1992. Cells in the marginal zone of the spleen. Int. Rev. Cytol. 132: 31-74. [Medline]
  2. Steinman, R. M., M. Pack, K. Inaba. 1997. Dendritic cells in the T cell areas of lymphoid organs. Immunol. Rev. 156: 25-37. [Medline]
  3. Morelli, A. E., A. T. Larregina, W. J. Shufesky, A. F. Zahorchak, A. J. Logar, G. D. Papworth, Z. Wang, S. C. Watkins, L. D. Falo, Jr, A. W. Thomson. 2003. Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production. Blood 101: 611-620. [Abstract/Free Full Text]
  4. Balazs, M., F. Martin, T. Zhou, J. Kearney. 2002. Blood dendritic cells interact with splenic marginal zone B cells to initiate T independent immune responses. Immunity 17: 341-352. [Medline]
  5. Attanavanich, K., J. F. Kearney. 2004. Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells. J. Immunol. 172: 803-811. [Abstract/Free Full Text]
  6. Lopes-Carvalho, T., J. F. Kearney. 2004. Development and selection of marginal zone B cells. Immunol. Rev. 197: 192-205. [Medline]
  7. Elomaa, O., M. Kangas, C. Sahlberg, J. Tuukkanen, R. Sormunen, A. Liakka, I. Thesleff, G. Kraal, K. Tryggvason. 1995. Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages. Cell 80: 603-609. [Medline]
  8. Geijtenbeek, T. B., P. C. Groot, M. A. Nolte, S. J. van Vliet, S. T. Gangaram-Panday, G. C. van Duijnhoven, G. Kraal, A. J. van Oosterhout, Y. van Kooyk. 2002. Marginal zone macrophages express a murine homologue of DC-SIGN that captures blood-borne antigens in vivo. Blood 100: 2908-2916. [Abstract/Free Full Text]
  9. Kang, Y. S., J. Y. Kim, S. A. Bruening, M. Pack, A. Charalambous, A. Pritsker, T. M. Moran, J. M. Loeffler, R. M. Steinman, C. G. Park. 2004. The C-type lectin SIGN-R1 mediates uptake of the capsular polysaccharide of Streptococcus pneumoniae in the marginal zone of mouse spleen. Proc. Natl. Acad. Sci. USA 101: 215-220. [Abstract/Free Full Text]
  10. van der Laan, L. J., E. A. Dopp, R. Haworth, T. Pikkarainen, M. Kangas, O. Elomaa, C. D. Dijkstra, S. Gordon, K. Tryggvason, G. Kraal. 1999. Regulation and functional involvement of macrophage scavenger receptor MARCO in clearance of bacteria in vivo. J. Immunol. 162: 939-947. [Abstract/Free Full Text]
  11. Kumamoto, Y., N. Higashi, K. Denda-Nagai, M. Tsuiji, K. Sato, P. R. Crocker, T. Irimura. 2004. Identification of sialoadhesin as a dominant lymph node counter-receptor for mouse macrophage galactose-type C-type lectin 1. J. Biol. Chem. 279: 49274-49280. [Abstract/Free Full Text]
  12. van den Berg, T. K., J. J. Breve, J. G. Damoiseaux, E. A. Dopp, S. Kelm, P. R. Crocker, C. D. Dijkstra, G. Kraal. 1992. Sialoadhesin on macrophages: its identification as a lymphocyte adhesion molecule. J. Exp. Med. 176: 647-655. [Abstract/Free Full Text]
  13. Jones, C., M. Virji, P. R. Crocker. 2003. Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake. Mol. Microbiol. 49: 1213-1225. [Medline]
  14. Dingjan, G. M., A. Maas, M. C. Nawijn, L. Smit, J. S. Voerman, F. Grosveld, R. W. Hendriks. 1998. Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton’s tyrosine kinase. EMBO J. 17: 5309-5320. [Medline]
  15. Nolte, M. A., R. Arens, M. Kraus, M. H. van Oers, G. Kraal, R. A. van Lier, R. E. Mebius. 2004. B cells are crucial for both development and maintenance of the splenic marginal zone. J. Immunol. 172: 3620-3627. [Abstract/Free Full Text]
  16. Karlsson, M. C., R. Guinamard, S. Bolland, M. Sankala, R. M. Steinman, J. V. Ravetch. 2003. Macrophages control the retention and trafficking of B lymphocytes in the splenic marginal zone. J. Exp. Med. 198: 333-340. [Abstract/Free Full Text]
  17. Thomas, C. A., Y. Li, T. Kodama, H. Suzuki, S. C. Silverstein, J. El Khoury. 2000. Protection from lethal gram-positive infection by macrophage scavenger receptor-dependent phagocytosis. J. Exp. Med. 191: 147-156. [Abstract/Free Full Text]
  18. Suzuki, H., Y. Kurihara, M. Takeya, N. Kamada, M. Kataoka, K. Jishage, O. Ueda, H. Sakaguchi, T. Higashi, T. Suzuki, et al 1997. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 386: 292-296. [Medline]
  19. van der Laan, L. J., M. Kangas, E. A. Dopp, E. Broug-Holub, O. Elomaa, K. Tryggvason, G. Kraal. 1997. Macrophage scavenger receptor MARCO: in vitro and in vivo regulation and involvement in the anti-bacterial host defense. Immunol. Lett. 57: 203-208. [Medline]
  20. Dijkstra, C. D., E. Van Vliet, E. A. Dopp, A. A. van der Lelij, G. Kraal. 1985. Marginal zone macrophages identified by a monoclonal antibody: characterization of immuno- and enzyme-histochemical properties and functional capacities. Immunology 55: 23-30. [Medline]
  21. Kraal, G., M. Janse. 1986. Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody. Immunology 58: 665-669. [Medline]
  22. van Rooijen, N., N. Kors, G. Kraal. 1989. Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination. J. Leukocyte Biol. 45: 97-104. [Abstract]
  23. Pikkarainen, T., A. Brannstrom, K. Tryggvason. 1999. Expression of macrophage MARCO receptor induces formation of dendritic plasma membrane processes. J. Biol. Chem. 274: 10975-10982. [Abstract/Free Full Text]
  24. Ito, S., M. Naito, Y. Kobayashi, H. Takatsuka, S. Jiang, H. Usuda, H. Umezu, G. Hasegawa, M. Arakawa, L. D. Shultz, et al 1999. Roles of a macrophage receptor with collagenous structure (MARCO) in host defense and heterogeneity of splenic marginal zone macrophages. Arch. Histol. Cytol. 62: 83-95. [Medline]
  25. Amlot, P. L., A. E. Hayes. 1985. Impaired human antibody response to the thymus-independent antigen, DNP-Ficoll, after splenectomy. Implications for post-splenectomy infections. Lancet 1: 1008-1011. [Medline]
  26. Gopal, V., A. L. Bisno. 1977. Fulminant pneumococcal infections in "normal" asplenic hosts. Arch. Intern. Med. 137: 1526-1530. [Abstract/Free Full Text]
  27. Garg, M., B. Subbarao. 1992. Immune responses of systemic and mucosal lymphoid organs to Pnu-Imune vaccine as a function of age and the efficacy of monophosphoryl lipid A as an adjuvant. Infect. Immun. 60: 2329-2336. [Abstract/Free Full Text]
  28. Shapiro, E. D., A. T. Berg, R. Austrian, D. Schroeder, V. Parcells, A. Margolis, R. K. Adair, J. D. Clemens. 1991. The protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N. Engl. J. Med. 325: 1453-1460. [Abstract]
  29. Perlmutter, R. M., D. Hansburg, D. E. Briles, R. A. Nicolotti, J. M. Davie. 1978. Subclass restriction of murine anti-carbohydrate antibodies. J. Immunol. 121: 566-572. [Abstract/Free Full Text]
  30. Sarvas, H. O., L. M. Aaltonen, F. Peterfy, I. J. Seppala, O. Makela. 1983. IgG subclass distributions in anti-hapten and anti-polysaccharide antibodies induced by haptenated polysaccharides. Eur. J. Immunol. 13: 409-414. [Medline]
  31. Shapiro, D. A., D. S. Threadgill, M. J. Copfer, D. A. Corey, T. L. McCool, L. L. McCormick, T. R. Magnuson, N. S. Greenspan, J. R. Schreiber. 1998. {gamma}3 Gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines. J. Immunol. 161: 3393-3399. [Abstract/Free Full Text]
  32. Hughes, D. A., I. P. Fraser, S. Gordon. 1995. Murine macrophage scavenger receptor: in vivo expression and function as receptor for macrophage adhesion in lymphoid and non-lymphoid organs. Eur. J. Immunol. 25: 466-473. [Medline]
  33. Pasparakis, M., S. Kousteni, J. Peschon, G. Kollias. 2000. Tumor necrosis factor and the p55TNF receptor are required for optimal development of the marginal sinus and for migration of follicular dendritic cell precursors into splenic follicles. Cell. Immunol. 201: 33-41. [Medline]
  34. Weih, D. S., Z. B. Yilmaz, F. Weih. 2001. Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines. J. Immunol. 167: 1909-1919. [Abstract/Free Full Text]
  35. Franzoso, G., L. Carlson, T. Scharton-Kersten, E. W. Shores, S. Epstein, A. Grinberg, T. Tran, E. Shacter, A. Leonardi, M. Anver, et al 1997. Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions. Immunity 6: 479-490. [Medline]
  36. Poljak, L., L. Carlson, K. Cunningham, M. H. Kosco-Vilbois, U. Siebenlist. 1999. Distinct activities of p52/NF-{kappa}B required for proper secondary lymphoid organ microarchitecture: functions enhanced by Bcl-3. J. Immunol. 163: 6581-6588. [Abstract/Free Full Text]
  37. Pabst, O., R. Forster, M. Lipp, H. Engel, H. H. Arnold. 2000. NKX2.3 is required for MAdCAM-1 expression and homing of lymphocytes in spleen and mucosa-associated lymphoid tissue. EMBO J. 19: 2015-2023. [Medline]
  38. Tumanov, A., D. Kuprash, M. Lagarkova, S. Grivennikov, K. Abe, A. Shakhov, L. Drutskaya, C. Stewart, A. Chervonsky, S. Nedospasov. 2002. Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues. Immunity 17: 239-250. [Medline]
  39. Kraal, G., H. Ter Hart, C. Meelhuizen, G. Venneker, E. Claassen. 1989. Marginal zone macrophages and their role in the immune response against T-independent type 2 antigens: modulation of the cells with specific antibody. Eur. J. Immunol. 19: 675-680. [Medline]
  40. van den Eertwegh, A. J., J. D. Laman, M. M. Schellekens, W. J. Boersma, E. Claassen. 1992. Complement-mediated follicular localization of T-independent type-2 antigens: the role of marginal zone macrophages revisited. Eur. J. Immunol. 22: 719-726. [Medline]
  41. Guinamard, R., M. Okigaki, J. Schlessinger, J. V. Ravetch. 2000. Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response. Nat. Immunol. 1: 31-36. [Medline]
  42. Claassen, E., N. Kors, N. Van Rooijen. 1986. Influence of carriers on the development and localization of anti-2,4,6-trinitrophenyl (TNP) antibody-forming cells in the murine spleen. II. Suppressed antibody response to TNP-Ficoll after elimination of marginal zone cells. Eur. J. Immunol. 16: 492-497. [Medline]
  43. Claassen, E., A. Ott, W. J. Boersma, C. Deen, M. M. Schellekens, C. D. Dijkstra, N. Kors, N. Van Rooijen. 1989. Marginal zone of the murine spleen in autotransplants: functional and histological observations in the response against a thymus-independent type 2 antigen. Clin. Exp. Immunol. 77: 445-451. [Medline]
  44. Garg, M., W. Luo, A. M. Kaplan, S. Bondada. 1996. Cellular basis of decreased immune responses to pneumococcal vaccines in aged mice. Infect. Immun. 64: 4456-4462. [Abstract]
  45. Martin, F., A. M. Oliver, J. F. Kearney. 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14: 617-629. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
N. J. Rogers, M. J. Lees, L. Gabriel, E. Maniati, S. J. Rose, P. K. Potter, and B. J. Morley
A Defect in Marco Expression Contributes to Systemic Lupus Erythematosus Development via Failure to Clear Apoptotic Cells
J. Immunol., February 15, 2009; 182(4): 1982 - 1990.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
M. P. Hubner, B. Pasche, S. Kalaydjiev, P. T. Soboslay, A. Lengeling, H. Schulz-Key, E. Mitre, and W. H. Hoffmann
Microfilariae of the Filarial Nematode Litomosoides sigmodontis Exacerbate the Course of Lipopolysaccharide-Induced Sepsis in Mice
Infect. Immun., April 1, 2008; 76(4): 1668 - 1677.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
F. Wermeling, Y. Chen, T. Pikkarainen, A. Scheynius, O. Winqvist, S. Izui, J. V. Ravetch, K. Tryggvason, and M. C.I. Karlsson
Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus
J. Exp. Med., October 1, 2007; 204(10): 2259 - 2265.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
F. Guo, D. Weih, E. Meier, and F. Weih
Constitutive alternative NF-{kappa}B signaling promotes marginal zone B-cell development but disrupts the marginal sinus and induces HEV-like structures in the spleen
Blood, October 1, 2007; 110(7): 2381 - 2389.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. Jozefowski, T. H. Sulahian, M. Arredouani, and L. Kobzik
Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides
J. Leukoc. Biol., October 1, 2006; 80(4): 870 - 879.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Y.
Right arrow Articles by Tryggvason, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Y.
Right arrow Articles by Tryggvason, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS