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

TryggvasonSoininen, Tatsuhiko Kodama, Georg Kraal and Karl Yunying Chen, Timo Pikkarainen, Outi Elomaa, Raija

http://www.jimmunol.org/content/175/12/8173doi: 10.4049/jimmunol.175.12.8173

2005; 175:8173-8180; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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

Yunying Chen,* Timo Pikkarainen,* Outi Elomaa,* Raija Soininen,† Tatsuhiko Kodama,‡

Georg Kraal,§ and Karl Tryggvason2*

The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by themarginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritonealmacrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of thescavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organizationof 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 moreapparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appear-ance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a cleardelay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the varioussubsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challengedwith a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockoutmice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being importantscavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction withendogenous ligands. The Journal of Immunology, 2005, 175: 8173–8180.

T he marginal zone (MZ)3 of the spleen has a central rolefor the initiation of both innate and adaptive immunityagainst blood-borne pathogens. Directly surrounding the

white pulp, the lymphoid compartment of the spleen, the MZ con-tains cells that are involved in both thymic-independent and -de-pendent immune responses (1). Among them are dendritic cellsthat can process Ags in the MZ, leading to activation and expres-sion of appropriate chemokine receptors, so that the dendritic cellswill enter the white pulp and can activate recirculating T cells toensure efficient local immune responses (2–4). In addition, the MZharbors a population of activated B cells, the MZ B cells, that

preferably locate here and are programmed to initiate a fast andintense Ab response to blood-borne viral and bacterial agents,making them key players in the early response to pathogens in thebloodstream (5, 6).

The most predominant cell population of the MZ is a specialtype of macrophage, characterized by specific expression of thepattern recognition receptors macrophage receptor with a collag-enous structure (MARCO) and specific intracellular adhesion mol-ecule-grabbing nonintegrin receptor 1 (SIGNR1), which have beenshown to bind a broad range of microbial Ags (7–10). This fact,together with their strategic position in the bloodstream of the MZmakes these macrophages important for efficient removal of patho-gens, but it may also lead to a concentration of pathogenic materialwhich can promote activation of macrophages and the MZ B cells,thus combining innate and adaptive immune responses. The mar-ginal zone macrophage (MZM) is distinct from the marginal met-allophilic macrophage (MMM) located at the inner face of the MZsinus, 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 micro-bial polysaccharides, in addition to playing a role in adhesive in-teractions with other leukocytes (11–13).

It has recently become clear that both B cells and the MZ mac-rophages are indispensable for the integrity and proper function ofthe MZ. In the absence of B cells during ontogeny, both theMMMs and the MZMs, as well as mucosal addressin cellular ad-hesion molecule-1 (MAdCAM-1)-positive sinus-lining cells in theMZ sinus are absent (14). But also the integrity and function of anestablished MZ was dependent on the presence of B cells, asshown in models where B cells could be depleted by removal ofthe BCR subunit Ig� and in a transgenic model in which all B cellswere gradually depleted due to overexpression of the TNF family

*Division of Matrix Biology, Department of Medical Biochemistry and Biophysics,Karolinska Institute, Stockholm, Sweden; †Department of Medical Biochemistry,Biocenter Oulu, University of Oulu, Oulu, Finland; ‡Department of Molecular Biol-ogy and Medicine, Research Center for Advanced Science and Technology, Univer-sity of Tokyo, Tokyo, Japan; and §Department of Cell Biology, Free University,Amsterdam, The Netherlands

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

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 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 andthe Foundation for Strategic Research.2 Address correspondence and reprint requests to Dr. Karl Tryggvason, Department ofMedical Biochemistry and Biophysics, Division of Matrix Biologi, Karolinska Insti-tutet, Scheeles Vag 2, B1, plan 4, S-17177 Stockholm, Sweden. E-mail address:karl.tryggvason@mbb.ki.se3 Abbreviations used in this paper: MZ, marginal zone; DAPI, 4�,6�-diamidino-2-phenylindole; KO, knockout; MAdCAM-1, mucosal addressin cellular adhesion mol-ecule-1; MARCO, macrophage receptor with a collagenous structure; M-CSF, mono-cyte-colony stimulating factor; MMM, marginal metallophilic macrophage; MZ,marginal zone, MZM, marginal zone macrophage; pp, postpartum; PS, polysaccha-ride; Siglec-1, sialic acid-binding Ig-like lectin-1; SIGNR1, specific intracellular ad-hesion molecule-grabbing nonintegrin receptor-1; SR-A, scavenger receptor A; TI-2,thymus-independent type 2.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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member CD70 (15). In contrast, the MZMs are essential for re-tention and trafficking of the MZ B cells, as shown by selectivedisruption of the inositol phosphatase SHIP (16). Interestingly, itwas demonstrated in the same study that the scavenger receptorMARCO showed activity for endogenous ligands on the MZ Bcells and was associated with the retention of the B cells in theMZ (16).

MARCO has a highly restricted expression pattern in adult miceliving under pathogen-free conditions (7). Besides the MZMs, it isconstitutively expressed in macrophages in the medullary cord oflymph nodes and in peritoneal macrophages. However, followingbacterial infection, MARCO is readily induced in other macro-phage populations such as in the spleen and liver (10). In contrast,the related scavenger receptor SR-A is normally widely expressedin macrophages, and it can be found in macrophages in the MZ andthe red pulp. Scavenger receptor A (SR-A) null mice have beenfound to be more susceptible than control mice to infection withListeria monocytogenes and Staphylococccus aureus, indicating arole for SR-A in antimicrobial host-defense mechanisms (17, 18).

In this study, we have analyzed the role of MARCO and SR-Ain the formation and function of the spleen using gene-targetedmice.

Materials and MethodsAnimals

The generation of MARCO�/� mice will be described elsewhere. Thesemice, as well as SRA�/� mice (18), were backcrossed to the C57BL/6 (B6)strain for �10 generations. To generate mice deficient for both MARCOand SR-A (double-KO), homozygous MARCO�/� mice were mated withhomozygous SR-A�/� mice. The resulting double heterozygotes (F1) werethen intercrossed to generate F2 mice. Double-KO F2 generation mice werethen mated together to produce mice used for the experiments described inthis study. To propagate the double-KO colony, the mice were backcrossedto the B6 strain. As controls, wild-type mice from breedings of theMARCO�/� mice were used. All mutant mice appeared normal and fertilein a pathogen-free environment. All mice studies were approved by theinstitutional review committee.

Tissue stainings and immunofluorescence microscopy

Fresh OCT-embedded tissues were frozen in liquid nitrogen and stored at�70°C. Cryosections of 8 �m were fixed in acetone for 10 min. Afterincubation in 10% normal serum from the species in which the secondaryAb was generated, the sections were incubated with the primary Ab, fol-lowed by several washes in PBS and incubation with a fluorescently la-beled secondary Ab. For double staining, tissue sections were first stainedfor one of the Ags, then incubated in 20% normal rat serum again, andsubsequently stained for the other Ag by first incubating with a biotinylatedmAb and then with fluorescently labeled streptavidin.

The following rat-anti-mouse mAbs were used: ED31, an anti-MARCOAb (19); ERTR9, which recognizes the C-type lectin SIGNR1 expressed inthe MZ (20); MOMA-1, recognizing a recently identified Siglec-1 Ag inthe 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, biotinyl-ated rabbit anti-mouse IgM (�-chain specific; Zymed Laboratories)was used.

Binding of the unconjugated primary Abs was detected with AlexaFluor 488- or 546-conjugated goat anti-rat Abs (Molecular Probes). Bio-tinylated primary Abs were detected with Alexa Fluor 594-conjugatedstreptavidin (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 DMEMcontaining 10% FCS or with PBS. Thioglycolate-elicited macrophageswere 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 typesby exploiting their ability to strongly adhere to glass or plastic. Cells wereplated in DMEM/FCS for 2 h on glass coverslips, after which unattachedand loosely adherent cells were removed by washing extensively with PBS.

The spreading of the macrophages was observed under phase-contrast mi-croscopy. Cells were fixed at indicated time points in 4% paraformalde-hyde, and stained with the Abs as described above. Actin cytoskeleton wasvisualized by staining with rhodamine-conjugated phalloidin (MolecularProbes). Cell nuclei were stained by 4�,6�-diamidino-2-phenylindole(DAPI) nucleic acid stain (Molecular Probes).

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

In some assays, equal volumes of peritoneal lavage fluid were plated on24-well culture plates for 2 h, and adherent cells were quantitated, after firstremoving nonadherent cells by several washes with PBS. For quantitation,the cells were fixed with glutaraldehyde, stained with crystal violet, rinsedwith water, and solubilized in 2% SDS. The absorbances were measured at595 nm.

Migration assay

Migration activity of resident peritoneal macrophages was assayed usingthe Transwell two-chamber system (Costar, 8-�m pore size, 6.5-mm insertdiameter). Resident peritoneal cells were harvested with PBS, washed oncewith DMEM containing 0.2% BSA and 15 mM HEPES (pH 7.4), andresuspended at 0.5 � 106 cells/ml in the same medium. One hundred mi-croliters of a cell suspension was applied into the upper chamber. Thelower chamber contained 10% FCS in DMEM (600 �l). After incubationfor 5 h at 37°C, cells on the upper side of the membrane were removed witha cotton tip and three rinses with PBS. Cells on the underside of the mem-brane were fixed with methanol overnight at 4°C, and stained with F4/80and DAPI. The membranes were mounted on glass slides, and cells werecounted under a fluorescence microscope with a �20 objective.

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

Macrophage depletion from the spleen

Liposome-entrapped dichloromethylene diphosphonate (clodronate) sus-pension was obtained from Clodronateliposomes (Free University). To de-plete cells from the spleen, 0.2-ml aliquots of the suspension were injectedi.v. into each mouse (22). Reappearance of macrophages and the other MZcell populations was monitored 4, 8, 11, 16, 21, 35, and 67 days after thetreatment, by staining frozen sections with the various Abs describedabove. 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 �g of thecapsular 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 AventisPasteur. A 500-�l sample of the vaccine diluted 1/25 in 0.9% NaCl wasinjected i.p. to 5–9 mice per genotype in each experiment. The experimentwas performed twice. Blood samples were taken by puncturing the tailartery at days 0, 7, 14, and 63 after the immunization from the same miceover time. Sera were stored at �20°C until analyzed. Serum anti-Pneumo23 IgM and IgG3 Igs were measured by ELISA. ELISA plateswere coated with 100 �l of the vaccine at the concentration of 2.3 �g/mlPBS 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 �l of the serum dilution (diluted in PBS/0.05% Tween 20)containing 0.5% BSA) was applied, and the anti-vaccine Abs were allowedto bind for 2 h at 37°C or overnight at 4°C. When testing anti-vaccine IgMIgs, we used a serum dilution 1/1000. In case of anti-vaccine IgG3, a serumdilution of 1/10 was used. These were found to be the proper dilutions inpilot experiments. Each serum sample was tested in duplicate. All bleed-ings were tested simultaneously. After the incubations, the wells werewashed several times with PBS/Tween 20 and incubated with biotinylatedrabbit anti-mouse IgM (Zymed Laboratories) or biotinylated monoclonalrat anti-mouse IgG3 (BD Pharmingen) for 90 min at 37°C. After washingswith PBST, the wells were incubated in HRP-conjugated streptavidin(Pierce; diluted 1/10,000 in PBST) at room temperature for 20 min, and

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then washed again. A substrate solution sample of 100 �l, a 1:1 mixture ofreagent A (H2O2) and reagent B (tetramethylbenzidine) (R&D Systems),was added into each well, and the color was allowed to develop for 10–20min. The reaction was terminated by adding 50 �l of 2 N H2SO4. Absor-bance values were read in a microplate reader at 450 nm and corrected byvalues obtained at 570 nm.

ResultsEffects of loss of MARCO and SR-A on spleen marginal zonemacrophages

We were interested in finding out whether the loss of MARCO, orboth MARCO and SR-A, affects the microarchitecture of thespleen MZ. The anti-SIGNR1 mAb ERTR9 was used to visualizethe MZMs, since these cells, with only minor exceptions, displayboth MARCO and SIGNR1 in normal adult wild-type mice (7, 19).Staining of the different genotypes for SIGNR1 revealed that thelack of MARCO, or both MARCO and SR-A, indeed resulted indefects in the MZ microarchitecture (Fig. 1). The SIGNR1-ex-pressing cells that, in wild-type mice, form a distinct zone of threeto four cell layers of macrophages between the red and white pulpcompartments showed a more diffuse staining pattern in theMARCO-KO and the double-KO mice. Fewer numbers ofSIGNR1-positive cells were found in the KO mice than in theirwild-type controls, and the cells were not tightly adhered to eachother or the MZ sinus, resulting in a more discontinuous MZ anda “gap” between the MZ macrophage layer and the sinus. Thisphenotype was even more severe in the double-KO mouse, indi-cating that both MARCO and SR-A are needed for the intactnessof the MZ macrophage layer. Indeed, in mice lacking only SR-A,the number of SIGNR1-expressing cells was clearly reduced butnot to the extent as found in the MARCO-KO and the double-KOmice, and the cells also showed more adherence to each other andthe marginal sinus. That the reduction in numbers of the SIGNR1-positive cells in the MZ of the KO mice was due to a reduction inactual cell numbers and not to that of SIGNR1 expression wasverified 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 re-duction in numbers of the MZMs was particularly evident in thecase of the double-KO mice.

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

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

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

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

Expression of SIGNR1 and Siglec-1 started much later than thatof MARCO or MAdCAM-1 during the neonatal spleen develop-ment, and they were not detected before day 7, the day when theMARCO- and MAdCAM-1-expressing cells already formed aclear MZ pattern. However, a more continuous rim-like MZ stain-ing 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- andSiglec-1-positive MZ macrophage populations. Thus, as seen inFig. 3C, at postnatal day 12, when a typical MZ pattern forSIGNR1 and Siglec-1 was found in wild-type mice, the expressionof SIGNR1 and Siglec-1 was just starting in the MARCO-KO

FIGURE 1. Genetic ablation of MARCO (MARCO-KO), SR-A (SR-A-KO) or both of these scavenger receptors (double-KO), alters the num-ber and microarchitecture of the MZMs in adult spleen. The MZMs (green)were identified with a mAb recognizing SIGNR1, a lectin-like transmem-brane protein. The endothelial cell layer lining the marginal sinus (red) wasstained with a mAb against MAdCAM-1. As compared with wild-typemice, there are fewer numbers of MZMs in the single-KO mice and evenfewer MZMs in mice deficient for both receptors. Note also the moresparse distribution of the MZMs in the KO mice.

FIGURE 2. Decreased number of MZMs in the double-KO mice as de-termined by staining for acid phosphatase activity. Spleen cryosectionsfrom wild-type and the double-KO mice were stained for this intracellularenzyme, a pan-macrophage marker that is also expressed in the MZMs atsignificant levels. As can be observed, the number of positive cells in theMZ is clearly lower in the double-KO than in wild-type mice.

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mice. Even less signal was detected in the double-KO mice. Incase of each genotype, the staining pattern detected at about day 20corresponded to that seen in adult mice (Fig. 1).

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

Considering the apparent differences in the appearance of the var-ious macrophage populations during the spleen development whenMARCO and SR-A are not expressed, we wished to explorewhether the rehabitation of splenic macrophages from bone mar-row precursor monocytes is also affected. For that purpose, weinjected clodronate liposomes i.v. into adult wild-type and KOmice, which leads to a rapid depletion (within 24 h) of all spleenred pulp and MZ macrophages, while macrophages in the whitepulp stay intact (22). Such depletion is normally followed by adifferential renewal of the splenic macrophages from blood mono-cytes, so that the red pulp macrophages reappear within 1 wk andthe MZ cells later. The transient absence of the macrophages alsoleads to the temporary disappearance of the MZ B cells and to theloss of MAdCAM-1 expression.

Following liposome administration, completeness of the mac-rophage depletion was confirmed by staining spleen sections forF4/80 (data not shown). Examination of wild-type mice 8 daysafter the liposome-treatment revealed that F4/80- and MARCO-positive cells had started to reappear in the red pulp. The numberof the MARCO-positive cells increased in the red pulp during thefollowing days, and the first of them started to localize to the MZaround 16 days after the depletion. At around day 21 the typicalring-like MZ MARCO-staining became obvious, and there wereonly some MARCO-positive cells left in the red pulp. At day 35the MARCO-staining pattern was indistinguishable from that seenin untreated mice (Fig. 4A).

As was the case with the MZ development during ontogeny,formation of the MARCO-positive MZ preceded the expression ofSIGNR1 and Siglec-1 during the recovery process after the lipo-some-treatment. Furthermore, similarly to the situation in ontog-eny, SIGNR1 and Siglec-1 appeared during this recovery processonly in the MZ. Siglec-1 appeared somewhat earlier than SIGNR1and was detected already at day 16 after the liposome-treatment.SIGNR1 expression first appeared at day 21 (data not shown).

FIGURE 3. Neonatal develop-ment of the spleen MZ. A, Staining ofspleen sections from wild-type miceindicates that MARCO- (red) andMAdCAM-1-expressing cells (green)are dispersed throughout the spleen atthe day of birth (0 pp). At day 3 pp,both cell populations are already con-centrating in the interface of the redand white pulp. A clear MZ pattern isestablished during the next few days.B, Formation of the MAdCAM-1-positive marginal sinus in the KOmice lags behind that in wild-typemice. The results indicate that theformation of the sinus rim is delayedby �3 days in the MARCO-KO miceand by �6 days in the double-KOmice. C, Staining of neonatal day 12spleen sections for the MZ macro-phage markers SIGNR1 and Si-glec-1. This staining reveals that theappearance of the SIGNR1-express-ing MZMs and the Siglec-1-express-ing MMMs is markedly delayed inthe KO mice. After day 12 pp thenumber of SIGNR1- and Siglec-1-positive cells increases in all threegenotypes, but the number ofSINGR1-positive cells in the KOmice never reaches that in wild-typemice (as shown in Fig. 1).

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Compared with wild-type mice, liposome depletion in the KOmice led to a significant delay in the reappearance of the MZ mac-rophage populations. The expression of MAdCAM-1 was also se-verely affected. As exemplified in Fig. 4B, at day 35 after thetreatment, when the typical MZ MARCO-staining pattern is de-tected (Fig. 4A), staining for SIGNR1 and Siglec-1 revealed a clearMZ staining pattern in wild-type mice, but not in the MARCO-KOand the double-KO mice (Fig. 4B). In these two KO strains,SIGNR1 and Siglec-1 expression was only starting to appear. Sur-prisingly, in contrast to the situation in ontogeny where no effects

of MARCO were seen on the MZ B cells, the reappearance ofthese cells after the liposome-treatment was severely affected inthe KO mice. The MZ B cells were found to have started reap-pearing 16 days after the depletion in wild-type mice but not in theKO mice. The situation at day 35 is shown in Fig. 4B. At the latesttime point tested (67 days after the depletion), the replenishment ofall MZ cell populations had reached in all three genotypes similarlevels as in their untreated counterparts (data not shown).

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

The absence of MARCO leads to cellular changes

The defects found in the architecture of the MZ in theMARCO-KO and the double-KO mice could be attributed to de-fects in adhesion or spreading of the macrophages in the absenceof MARCO and SR-A proteins. In studies with peritoneal macro-phages from SR-A KO mice, defects in their spreading propertieshave been reported (18). Because ectopic expression of MARCOcan induce formation of long dendritic processes in different celllines (23), we examined the spreading properties of macrophagesfrom the MARCO-KO and the double-KO mice. When plated onglass coverslips in the presence of serum for 24 h, spreading of theresident peritoneal macrophages from the MARCO- and dou-ble-KO mice was significantly impaired compared with the corre-sponding wild-type cells (Fig. 5, C–F), which constitutively ex-press MARCO (Fig. 5A) (and SR-A). We also consistentlyobserved that smaller numbers of resident peritoneal macrophagescould be recovered from the MARCO-KO and double-KO micethan from wild-type mice (Fig. 5G). To see whether MARCOplays a role in the recruitment of new macrophages to the perito-neum, influx was induced using the thioglycollate-induced perito-nitis model. However, the newly recruited macrophages did notexpress MARCO (Fig. 5B). This might be because the recruitedcells are not mature macrophages. Notably, neither was LPS (aloneor together with IFN-�) able to induce MARCO-expression in thiscell population in vivo or in vitro (data not shown), although itrapidly induces MARCO expression in many resident macrophagesubpopulations (10, 19, 24).

We then examined the migratory ability of the resident perito-neal macrophages from the double-KO and wild-type mice in atwo-chamber migration system in which the cells were allowed tomigrate for 5 h from serum-free medium toward medium contain-ing 10% FCS. Both cell populations exhibited low chemotacticresponsiveness, but in each of the several experiments we foundhigher numbers—2- to 3-fold or more—of the double-KO mac-rophages than their wild-type counterparts on the lower side of themembrane. These findings support the possibility that lack ofMARCO and SR-A affects the retention of macrophages in theperitoneal cavity.

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

The spleen plays a major role in protection against infections withencapsulated bacteria, such as S. pneumoniae (25–27). Protectionis mainly provided by Abs against capsular polysaccharides (PSs)

FIGURE 4. Reappearance of the different MZ cell populations after celldepletion by a single i.v. injection of clodronate-containing liposomes. A,The characteristic concentric MZ distribution pattern of MARCO can bedetected �15–20 days after the liposome treatment. When the spleen sec-tions are stained with the mAb ED31 recognizing the scavenger receptorcysteine-rich domain of MARCO 4 days after the liposome treatment, onlysome fibrous staining left from depleted cells can be detected (data notshown). 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 sig-nificantly at day 11. By day 16, significant numbers of the MARCO-pos-itive cells have already migrated to the MZ, and a MARCO-positive MZcan be distinguished. At day 21, the characteristic ring-like MZ distributionpattern of MARCO is very obvious. At day 35, ED31 gives a very similarstaining pattern to that seen in an untreated mouse. B, The different MZ cellpopulations appear in the KO mice with delayed kinetics as compared withsimilarly treated wild-type mice. The situation at day 35 after the liposometreatment is shown as an example. The panels represent the followingstainings: upper row, the MZM marker SIGNR1; middle row, the MMMmarker 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 theiruntreated control mice can be seen at the last time point examined, day 67.

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(28). Capsular PSs belong to the class of T-independent type 2 Agsagainst which the MZ with its macrophages and B cells plays animportant role (1). We therefore wished to compare the Ab re-sponses against a preparation of capsular PSs from 23 commonlyoccurring serotypes of pneumococcal bacteria in wild-type and theMARCO-KO and the double-KO mice. Because TI-2 Ags elicitprimarily production of IgM and IgG3 in mice (29–31), only thelevels of these two Ab classes were measured. Serum IgM andIgG3 levels were measured 1, 2, and 9 wk after injection of asingle dose of capsular PSs. The results for IgM are shown in Fig.6A, and represent at each time point the absorbance values ofpooled sera at one dilution. For each genotype, sera from 10–15mice immunized in two sets of injections were pooled. The resultsindicate higher anti-PS IgM response in wild-type mice than in theMARCO- or the double-KO mice. The double-KO mice showed avery low response, but these mice had higher anti-PS IgM levelsbefore immunization than the other genotypes. The levels of total

IgM Abs were also highest in this group (data not shown). In thecase of IgG3, there was a large degree of mouse-to-mouse vari-ability ranging from no response to high response even within eachgenotype. This was true particularly for the KO mice, in fact, whenbased on the median value virtually no response was seen in micedeficient in both MARCO and SR-A (Fig. 6B).

DiscussionMARCO and SR-A, two important scavenger receptors with abroad ligand specificity, play an important role in the recognitionof pathogens and the initiation of the innate immune activation.Interestingly, interaction with self-ligands may be involved in thelocalization and migration of cells expressing the receptors, as sug-gested from the effects of SR-A on adhesive and spreading prop-erties of macrophages, and from the fact that ectopic expression ofMARCO led to dramatic cell shape changes in transfected cells.

We therefore focused in this study on the effects of ablation ofMARCO and both MARCO and SR-A on the integrity of thespleen, especially since the MZMs uniquely express MARCO, butalso 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-typemice, and their number was reduced. These structural defects inthe MZ were even more severe in the double MARCO/SR-A-KOmice than in the MARCO-KO mice, indicating that both MARCOand SR-A are needed for the proper structural integrity of this area.

The ontogeny studies and the effects of liposome depletion re-vealed an interesting spatiotemporal relationship betweenMARCO and SIGNR1 expression. First, MARCO was found to beexpressed very early, much earlier than SIGNR1, in the spleens ofwild-type mice both during ontogeny and after the liposome-treat-ment, first appearing on the red pulp macrophages. Second, a sig-nificant delay in the appearance of SIGNR1 expression was found

FIGURE 5. Effects of MARCO and SR-A ablation on peritoneal mac-rophages. Resident (A) or thioglycollate-elicited (B) macrophages collectedfrom the peritoneal cavity of wild-type mice were cultured on glass cov-erslips for 24 h and stained with the mAb ED31 for MARCO (red). Allcells were visualized by nuclear DAPI staining (blue). Resident peritonealmacrophages express MARCO. Only 10% of the peritoneal macrophagescollected 4 days after the thioglycollate-injection were found to expressMARCO. Because the thioglycollate-treatment increases the size of theperitoneal macrophage population by �10-fold, this suggests that the elic-ited cells do not express MARCO. C–F, Resident peritoneal macrophagesfrom the different mouse strains were grown on coverslips for 24 h in thecomplete culture medium (C, wild-type; D, MARCO-KO; E, SR-A-KO; F,double-KO). After fixation with paraformaldehyde, cells were visualizedby staining for F-actin (red). These panels clearly demonstrate the defectivespreading properties of the KO cells. G, The percentage of F4/80-positivecells in the cytospun total peritoneal leukocyte population. The results in-dicate that the KO mice exhibit a marked reduction in the number of theresident peritoneal macrophages (p � 0.001 by Student’s t test).

FIGURE 6. Impaired responses against a pneumococcal polysaccharidevaccine in the KO mice. Wild-type and the different KO mice were im-munized i.p. with 25 �g of a 23-valent pneumococcal vaccine, Pneumo-wax, and serum anti-Pneumo23 IgM and IgG3 Ab levels were measured atthe indicated times. The results are shown in absorbances at one serumdilution. In the case of IgM (A), they represent at each time point theabsorbance values of pooled sera collected from mice immunized in twoindependent experiments (n: wild type, 15; MARCO-KO, 13; SR-A-KO,10; double-KO, 12). In the case of IgG3 (B), the median absorbance valuesfrom one of two similar experiment are shown. Each experimental groupconsisted of five to nine mice (‚, wt; �, MARCO-KO; �, SR-A-KO; E,double-KO).

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in the KO mice compared with similarly treated wild-type mice.Strikingly, SIGNR1 expression was not detected in the red pulpmacrophages, but only in the MZMs. This suggests that the ex-pression of MARCO (and possibly SR-A) is necessary for theproper development and organization of the MZ and precedes fur-ther differentiation of the MZMs as evidenced by the expression ofSIGNR1.

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

A possible mechanism for the structural defects in the MZ of thespleen could lie in the fact that MARCO is necessary for the properadhesion and retention of the cells within the MZ through inter-action with local structural components. This notion is supportedby our findings that peritoneal macrophages from theMARCO-KO mice show defects in adhesion and spreading, andpossibly also in cell retention, because the KO mice were found tohave significantly reduced numbers of resident peritoneal macro-phages. One possibility is that if the cells have defects in theiradhesion and spreading capabilities, their lifespan could be af-fected. In contrast, the results from the migration assays point tothe possibility that the efflux rate of macrophages out of the peri-toneum is higher in the KO mice. Further studies will be needed toinvestigate these possibilities.

Our findings on the effects of MARCO and SR-A are in linewith other reports showing that a complex interplay exists betweenthe various cell types found in the MZ, including the differentmacrophage subpopulations, B cells and even the stromal sinus-lining cells. In particular, it appears that genetic defects leading toabsence or low numbers of the MZMs coincide with defects in theMAdCAM-1-positive sinus-lining cells. For example, ablation ofsuch genes as those encoding TNF, p55TNF-R, RelB, and Bcl-3results in phenotypes where the numbers of the SIGNR1-positiveMZMs are reduced drastically, and MAdCAM-1 is either com-pletely missing or expressed at very low levels (33–36). Similarly,mice lacking the transcription factor NKX2.3, which is requiredfor MAdCAM-1 expression, were found to have reduced numbersof Siglec-1-positive MMMs, as well as drastically reduced num-bers of SIGNR1-positive macrophages (37). Furthermore, theselatter cells were not restricted any more to the MZ but were scat-tered in the red pulp.

In addition, there clearly is an interplay between B cells andmacrophages, which is necessary for the proper organization of theMZ, as well as for the retention and function of both of these cellpopulations. The absence of signaling through lymphotoxin � on Bcells led to a reduction in size of the MZ B cell-population, but alsoto that of the SIGNR1-, Siglec-1-, and MAdCAM-1-positive cellpopulations (38). Related to this finding, a recent study with sev-eral KO models revealed that B cells are not only important for thedevelopment but also for the maintenance of the MZ macrophagesubpopulations (15). Another recent study has shown that the in-teraction between MARCO and a cell surface determinant on theMZ B cells contributes to the retention of the MZ B cells withinthe MZ (16). This is in contrast to our ontogeny studies where theappearance of the MZ B cells showed no difference in the absenceof MARCO but is in line with our liposome depletion studies,which also argue for a role of MARCO in the retention of the MZB cells. It may well be the case that the importance of this inter-action for the MZ B cell retention is not uncovered in the chronic

loss-of-gene function situation, but only in an acute situation, suchas the recovery process after the liposome treatment.

We observed that the ablation of MARCO led to an impairedresponse against a TI-2-Ag. After i.v. injection, these types of Agsare rapidly captured in the spleen by the MZMs and the MZ B cellsvia, respectively, SIGNR1- and complement-mediated processes(39–41). A previous study with Pyk-2-deficient mice demon-strated an important role for the MZ B cells in the Ab responses toone type of TI-2-Ag, Ficoll (41), but the role of the MZMs in thisregard is controversial. In one study elimination of the MZM cellswith clodronate-containing liposomes resulted in a strong decreaseof the Ab response to TNP-Ficoll (42), but this could not be con-firmed in other studies, including those where the MZMs wereeliminated with the toxin gelonin coupled to the anti-SIGNR1 AbERTR9 (39, 43). In contrast, it is known that there is an age-associated decline in Ab responses to the pneumococcal PS inhumans 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 plasticor by passage through a Sephadex column, indicating a macro-phage-like nature of these cells. The exact role of the accessorycells in the vaccine response is not known, but they may have arole in the generation of a proper microenvironment for the MZ Bcells, because the response in the aged mice spleen cells could berestored by supplementation with a variety of cytokines (44). It isof interest to note in this regard that staining of spleen section fromaged mice for MARCO or SIGNR1 indicates significantly reducednumbers of MZMs compared with young adult mice (data notshown).

In addition to the MZ B cells, the peritoneal B1 B cells, themajor produces of natural Abs, also participate in TI-2-responses(4, 45). In particular, when mice are challenged i.p. with a lowdose of a TI-2-Ag, these cells are the first and probably the onlyexposed and responding B cell population (45). Notably, primedperitoneal macrophages have been found to effectively support B1cell differentiation (4). We did not see any differences in the sizeof B1 cell population between the genotypes (data not shown).However, it cannot be excluded that the reduction in the number ofperitoneal macrophages is one reason to the lowered TI-2 responsein the KO mice.

In summary, our data show that in addition to their role as clas-sical pattern recognition receptors involved in the uptake of patho-gens, MARCO and, to a lesser extent, SR-A, play a role in thestructural organization and cellular interactions of the marginalzone of the spleen, most likely through interactions with self li-gands, and that such interactions are crucial for the functioning ofthe spleen and an optimal homeostasis.

DisclosuresThe authors have no financial conflict of interest.

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