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The Journal of Immunology, 2003, 171: 1148-1155.
Copyright © 2003 by The American Association of Immunologists

Macrophages of the Splenic Marginal Zone Are Essential for Trapping of Blood-Borne Particulate Antigen but Dispensable for Induction of Specific T Cell Responses

Peter Aichele*,{dagger}, Jana Zinke*, Leander Grode*, Reto A. Schwendener{dagger},{ddagger}, Stefan H. E. Kaufmann1,* and Peter Seiler2,*

* Abteilung Immunologie, Max-Planck-Institut für Infektionsbiologie, Berlin, Germany; {dagger} Institut fur Medizinische Mikrobiologie Hygiene, Universitatsklinikum Freiburg, Freiburg, Germany; and {ddagger} Paul Scherrer Institute, Villingen, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rapid removal of pathogens from the circulation by secondary lymphoid organs is prerequisite for successful control of infection. Blood-borne Ags are trapped mainly in the splenic marginal zone. To identify the cell populations responsible for Ag trapping in the marginal zone, mice were selectively depleted of marginal zone macrophages and marginal metallophilic macrophages. In the absence of these cells, trapping of microspheres and Listeria monocytogenes organisms was lost, and early control of infection was impaired. Depletion of marginal zone macrophages and marginal metallophilic macrophages, however, did not limit Ag presentation because Listeria-specific protective T cell immunity was induced. Therefore, marginal zone macrophages and marginal metallophilic macrophages are crucial for trapping of particulate Ag but dispensable for Ag presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Efficient control of pathogens by the immune system is promoted by a highly organized microarchitecture of secondary lymphoid organs. These structures form the basis for trapping, transport, processing, and presentation of Ags, a prerequisite for initial constraint of pathogens and successful induction of specific immunity. The spleen is responsible for filtering/clearing blood borne particles. Microanatomically, the spleen is divided into white pulp and red pulp separated by the marginal zone. The marginal zone consists of sinus-lining reticular cells, marginal zone B cells, dendritic cells (DC), 3 marginal metallophilic macrophages (MM), and marginal zone macrophages (MZM). In the marginal zone, the blood leaves the terminal arterioles into open sinuses, the blood flow is slowed down, and blood-borne particles are trapped with high efficiency (1, 2, 3). Within the marginal zone, the MZM form a ring at the outer border of the sinus. They are large with long cellular processes, are tightly bound to the reticular meshwork, and appear to exhibit high phagocytic activity. MZM were first described by their ability to retain and internalize haptenated polysaccharides (4, 5). MM form a thin rim at the inner border of the marginal zone (6), show affinity for silver (metallophilic), and are less phagocytic than MZM (1, 7).

Trapping in the marginal zone has been shown for various soluble (5, 8, 9, 10, 11, 12) and particulate (6, 13, 14, 15, 16, 17) model Ags differing in size and surface characteristics, as well as for live pathogens with a more complex surface structure like Candida, Brucella, Lactobacillus, or vesicular stomatitis virus (18, 19, 20). Although blood-borne Ags are associated with MZM and MM early after application, it remained unclear whether these cell populations are crucial for the capacity of the marginal zone to trap Ag and whether the trapping/clearing functions of the marginal zone have an impact on the control of infection and the induction of acquired immunity.

Here we took advantage of an in vivo depletion protocol that allows selective elimination of MZM and MM using low dose clodronate liposomes (LCL), to evaluate the role of these cells in trapping of particulate Ags and control of Listeria monocytogenes infection. Our results 1) emphasize the importance of the marginal zone in early trapping of particulate Ags, 2) functionally link Ag trapping to MZM and MM, 3) demonstrate that reduced Ag trapping in the marginal zone impairs early control of infection due to exacerbated hematogenic spread of the pathogen, and 4) reveal that MZM and MM are dispensable for Ag presentation to induce protective T cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 and C57BL/6-Thy-1.1 mice were bred at our breeding facilities at the Bundesamt für Risikobewertung (Berlin, Germany) under specific pathogen-free conditions. Experiments were conducted under conventional housing conditions according to the German animal protection law.

Bacteria and determination of bacterial titers

L. monocytogenes strain EGD was originally obtained from G. B. Mackaness (Trudeau Institute, Saranac Lake, NY). Bacteria were cultured in tryptic soy broth (Difco, Detroit, MI) overnight at 37°C on a rocking platform. Bacteria were washed twice in PBS, diluted to the appropriate concentration, and injected i.v. in a volume of 200 µl. The infection dose was controlled by plating dilutions of the inoculum on tryptic soy broth agar plates. To determine the bacterial load of organs, mice were killed at the time points indicated. Organs were homogenized in 1 ml of PBS, and serial dilutions of the homogenates were plated on agar plates. Colonies were counted after incubation at 37°C for 24 h.

Preparation of clodronate liposomes

Liposomes were prepared as described earlier with minor modifications (21, 22, 23). In brief, soy phosphatidylcholine, DL-{alpha}-tocopherol, and cholesterol (all from Sigma-Aldrich, St. Louis, MO; 1:0.01:0.3 molar ratio) were dissolved in chloroform. After evaporation of the solvent, dry lipid film was dispersed in 10 ml of clodronate solution (Ostac, Roche, Switzerland) by careful shaking. Suspension was sonicated three times for 5 min and freeze-thawed in three cycles of liquid nitrogen and water at 37°C. Liposomes were washed twice in PBS (50,000 x g) to remove free clodronate. For LCL, final solution was adjusted to an OD600 of 1.2, and 200 µl were injected i.v. Mice either treated with empty liposomes or left untreated showed identical phenotypes and were used as controls.

Fluorescent particulate Ags

The fluorescent Fluoresbrite carboxy YG microspheres (50 nm diameter; Polysciences Europe, Eppelheim, Germany) were washed in PBS. For injection, stock solution was diluted 1/50 in PBS and 200 µl were administered i.v. into the tail vein. A 10-ml overnight culture of L. monocytogenes was washed twice in ice-cold PBS. Pellet was resuspended in 10 ml of PBS, and 100 µl of 0.5 mM CFSE (Molecular Probes, Eugene, OR) was added. After 10 min of incubation at 37°C, the staining reaction was stopped by adding 500 µl of FCS, and bacteria were washed three times in ice-cold PBS and adjusted to the appropriate concentration before i.v. injection. Spleens were removed 30 min postinfection and snap frozen in liquid nitrogen; localization of fluorescent particulate Ags was analyzed on cryosections using an epifluorescence microscope.

Immunohistochemistry

Freshly removed organs were immersed in Tissue-Tek O.T.C. (Miles Laboratories, Elkhart, IN), snap frozen in liquid nitrogen, cut into 5-µm-thick sections, air dried, and fixed in acetone for 10 min. These sections were incubated with primary rat Abs specific for: marginal zone macrophages (ERTR-9; Dianova, Hamburg, Germany), marginal metallophilic macrophages (MOMA-1; Biomedicals, Augst, Switzerland), red pulp macrophages (F4/80; ATCC HB-198), DC (anti-CD11c; BD PharMingen, San Jose, CA), B cells (rat anti-mouse IgM; Southern Biotechnologies, Birmingham, AL), and reticular endothelial cells (MECA-367; BD PharMingen). Primary rat mAbs were detected by a 2-fold sequential incubation with goat anti-rat Ig (Caltag Laboratories, Burlingame, CA) and alkaline phosphatase-conjugated donkey anti-goat Ig (Jackson ImmunoResearch Laboratories, West Grove, PA). Alkaline phosphatase was detected by incubation with naphthol AS-BI phosphate (Sigma-Aldrich) and New Fuchsin (Merck, Darmstadt, Germany) as substrate. Endogenous alkaline phosphatase activity was blocked by levamisole (Sigma-Aldrich). Sections were counterstained with Mayer’s hemalum (Merck).

Detection of Listeria-specific Abs

Collected sera were kept at -20°C until use. For ELISA, 96-well NUNC Maxisorb plates (Nalgene-NUNC, Naperville, IL) were incubated at 4°C overnight with 100 µl of 50x concentrated L. monocytogenes EGD supernatant diluted 1/2 in 0.5 M carbonate buffer, pH 9.6. After two washes with PBS-0.05% Tween, plates were blocked at 37°C for 2 h with 150 µl of 1% BSA in PBS. Individual sera were added to the samples starting at a dilution of 1/10 and serially diluted 1/2 in 0.5% BSA in PBS-Tween (dilution buffer) for determining the end point titers. After 1.5 h at 37°C, plates were washed three times, and 100 µl of alkaline phosphatase-coupled goat anti-mouse IgG diluted 1/1000 in dilution buffer were added. Plates were incubated at 37°C for 1 h and then washed four times. The colorimetric assay was developed with 50 µl of p-nitrophenyl phosphate (Sigma-Aldrich) in diethanolamine buffer, pH 9. The OD405 was determined with a SpectraMax 250 ELISA reader (MWG Biotech, Ebersberg, Germany) after 20 min. Each assay was performed in duplicates, and data represent means ± SD.

Cytokine ELISA

Collected sera were kept at -20°C until use. IFN-{gamma} was determined by double-sandwich ELISA using the mAb anti-IFN-{gamma} R4-2A2 and anti-IFN-{gamma} XMG1.2 Biotin (BD PharMingen), which recognize different epitopes of the cytokine. For ELISA, 96-well NUNC Maxisorb plates (Nalgene-NUNC) were incubated at 4°C overnight with 100 µl of the mAb R4-2A2 at a concentration of 1 µg/ml in 0.5 M carbonate buffer, pH 9.6. Individual sera were added starting at a dilution of 1/10 and serially diluted 1/2 in 0.5% BSA in PBS-Tween. After overnight incubation at 4°C, plates were washed three times, and 100 µl of mAb XMG1.2 Biotin (2 µg/ml) were added and incubated for 1 h at 37°C in a wet chamber. After further washing, 100 µl of alkaline phosphatase-coupled streptavidin diluted 1/1000 in dilution buffer was added. Plates were incubated at 37°C for 1 h and then washed four times. The colorimetric assay was developed with 50 µl of p-nitrophenyl phosphate (Sigma-Aldrich) in diethanolamine buffer, pH 9. The OD405 was determined with a SpectraMax 250 ELISA reader after 20 min. Each assay was performed in duplicates, and data represent means ± SD. Murine r-IFN-{gamma} from R&D Systems (Minneapolis, MN) with an activity of 107 U/mg was diluted in PBS, 0.1% BSA as standard.

Adoptive transfers

C57BL/6-Thy-1.1 mice, treated with LCL or empty liposomes, were infected i.v. with 103 CFU of L. monocytogenes; 25–30 days after primary infection, mice were killed, spleens were removed, and single-cell suspensions were prepared. Splenocytes (4 x 107) from LCL or control C57BL/6-Thy-1.1 mice were transferred into sex-matched, nonirradiated wild-type C57BL/6-Thy-1.2 mice in a volume of 0.5 ml. After 6 h, recipients were infected i.v. with 5 x 103 CFU of L. monocytogenes. FACS of blood samples from recipient mice was done at different time points after infection using FITC-conjugated anti-CD8 mAb (clone 53.6.7; BD PharMingen) and PE-conjugated Thy-1.1 mAb (CD90.1; clone OX-7; BD PharMingen).

Statistics

Survival rates were analyzed by the log rank test. Other data were evaluated by the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selective depletion of MZM and MM

C57BL/6 mice were treated with LCL. Three days later, spleens were removed and the tissue structures were evaluated on cryosections. MZM and MM were completely depleted in LCL-treated mice (Fig. 1, A and C) compared with controls (Fig. 1, B and D), whereas red pulp macrophages were not affected by this regimen (Fig. 1, E and F). Importantly, other cell types of the marginal zone, notably DC (Fig. 1, G and H), B cells (Fig. 1, I and K), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)+ reticular cells (Fig. 1, L and M) were not eliminated by LCL administration. Complete depletion of MZM and MM lasted for 6–8 days; thereafter, the marginal zone was slowly repopulated by the two macrophage populations within 15–20 days (data not shown). Control treatments using empty liposomes with the same lipid composition and concentration influenced neither the splenic microarchitecture nor the functional readouts described thereafter (data not shown). LCL treatment allowed selective elimination of MZM and MM without affecting other cells of the marginal zone or the organization of splenic follicular structures. Histological analysis of the liver revealed that Kupffer cells were not or only slightly reduced after LCL administration (Fig. 2). Comparable results were obtained for lung and kidney where tissue macrophages were not depleted (data not shown). In earlier studies, we have demonstrated that LCL treatment had no direct toxic effect on T and B cells (22).



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  		FIGURE 1. Selective depletion of splenic MZM and MM by LCL treatment. C57BL/6 mice received LCL i.v. or were left untreated. Three days later, spleens were removed and snap frozen in liquid nitrogen, and cryosections were stained with mAb specific for marginal zone macrophages (A and B), marginal metallophilic macrophages (C and D), red pulp macrophages (RPM; E and F), DC (G and H), B cells (I and K), and sinus-lining reticular cells (L and M). Original magnifications, x100 (A–H) and x200 (I–M). Representative sections from 10 mice.

 


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  		FIGURE 2. LCL treatment does not eliminate liver macrophages. C57BL/6 mice received LCL (A) or were left untreated (B). Three days later, livers were removed and snap frozen in liquid nitrogen, and cryosections were stained with mAb specific for Kupffer cells (F4/80). Original magnification x100. Representative sections from 10 mice.

 
Impaired Ag trapping of the marginal zone after selective depletion of MZM and MM

To evaluate the role of MZM/MM in trapping of blood-borne particles, the early splenic distribution of microspheres and L. monocytogenes after i.v. administration was analyzed in the presence or absence of MZM/MM. Thirty minutes after Ag application, both microspheres and listeriae were trapped nearly completely in the marginal zone of the spleen in control mice (Fig. 3, A and C). Weak fluorescence signals were detected in the red pulp and virtually none in the white pulp, indicating that only few particles escaped Ag trapping in the marginal zone. In mice selectively depleted of MZM/MM, microspheres and listeriae were disseminated into the red pulp and were no longer retained exclusively in the marginal zone (Fig. 3, B and D). Interestingly, neither marginal zone B cells and DC nor reticular cells could compensate for the lack of MZM/MM and failed to maintain the original trapping capacity of the marginal zone. Thus, our results functionally link Ag trapping in the marginal zone to MZM and MM.



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  		FIGURE 3. Loss of Ag-trapping capacity of the marginal zone after selective depletion of MZM and MM. C57BL/6 mice were treated i.v. with PBS (A and C) or LCL to deplete MZM and MM (B and D). Three days later, 200 µl of fluorescent microspheres (50 nm in diameter; A and B) or 5 x 106 CFU CFSE-labeled L. monocytogenes (C and D) were administered i.v. Thirty minutes later, spleens were removed and snap frozen in liquid nitrogen, and the local distribution of the particles on cryosections was analyzed by fluorescence microscopy. Original magnification x100. Representative sections from eight mice.

 
Exacerbated early hematogenic spread of L. monocytogenes to peripheral organs after selective depletion of MZM and MM

Resistance against L. monocytogenes critically depends on innate as well as acquired immunity (24, 25). In the early phase of infection, resident macrophages, neutrophils, and natural killer cells are involved in control of listeriae (26, 27, 28, 29, 30). Thereafter, sterile eradication of bacteria is achieved by CD8+ T cells and CD4+ T cells (31, 32).

To analyze early hematogenic spread of L. monocytogenes, MZM/MM-depleted and control mice were infected i.v. with a high dose of 107 CFU of bacteria, and the titers in different organs were determined 30 and 120 min later (Fig. 4A). In the absence of MZM/MM, bacterial titers were slightly elevated in the spleen. Bacterial burdens in livers, a preferential target organ for listeriae, did not differ 30 and 120 min postinfection in MZM/MM-depleted and control mice. Interestingly, 6 h after infection the bacterial load of the liver was reduced by 1 order of magnitude in both experimental groups (data not shown). This indicates that the characteristic early listeriocidal activity of the Kupffer cells was not impaired by LCL treatment. This is consistent with the fact that Kupffer cells are not eliminated and demonstrates that they are still functional. In contrast, listerial titers in kidney and lung, organs not preferentially targeted by listeriae, were significantly increased in MZM/MM-depleted mice. This is further illustrated by elevated levels of listeriae in the blood of MZM/MM-depleted mice early after high dose infection (Fig. 4B). Control treatments using empty liposomes with the same lipid composition and concentration led to the same results as in untreated animals. Thus, impaired Ag trapping in the marginal zone in the absence of MZM/MM resulted in enhanced hematogenic spread of listeriae to peripheral organs.



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  		FIGURE 4. Early hematogenic spread of L. monocytogenes in MZM/MM-depleted mice after high dose infection. A, C57BL/6 mice, LCL treated at day -3 (•) or left untreated ({circ}), were infected i.v. with 1 x 107 CFU of L. monocytogenes at day 0. Bacterial titers in spleen, liver, kidney, and lung were followed 30 and 120 min after infection. Shown are two experiments with individual values of three mice per group. B, Bacterial titers in the blood of LCL-treated ({blacksquare}) and control animals ({square}) were examined 4 and 24 h after i.v. infection with 103 or 108 CFU of L. monocytogenes. Dashed lines represent detection limit of the assay. *, p < 0.05

 
Impaired control of early L. monocytogenes infection after selective depletion of MZM and MM

To analyze the impact of reduced Ag trapping in the marginal zone and early hematogenic spread on host resistance, mice were infected with various doses of listeriae. In a first set of experiments, MZM/MM-depleted and untreated mice were infected with a sublethal dose of L. monocytogenes and survival of mice was monitored. As shown in Fig. 5, >90% of control animals survived, whereas natural resistance of MZM/MM-depleted mice was significantly decreased with only 20% of mice surviving infection (p < 0.001). In a second set of experiments, animals were infected with low dose L. monocytogenes, and bacterial titers in spleen, liver, kidney, and lung were determined (Fig. 6). In the absence of MZM/MM, bacterial titers were slightly increased in spleen and to a greater degree in liver between days 1 and 4 postinfection compared with controls. Bacteria were not detectable in kidney and lung of control mice after low dose infection, whereas the organs of MZM/MM-depleted animals harbored a significant bacterial burden. By day 7, mice of both experimental groups controlled low dose Listeria infection, and bacteria were eliminated by day 12. Hence, despite the selective depletion of MZM/MM, an efficient T cell response must have been induced leading to sterile eradication of listeriae.



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  		FIGURE 5. Impaired survival of mice after sublethal infection with L. monocytogenes in the absence of MZM and MM (MZM/MM-depl.). LCL-treated ({blacktriangleup}) or control ({triangleup}) C57BL/6 mice were infected i.v. with 2 x 104 CFU of L. monocytogenes, and survival of animals was followed (experimental groups are significantly different according to log rank test; p < 0.001). Data were pooled from two independent experiments with a total of 15 mice per group.

 


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  		FIGURE 6. Reduced early control of low dose L. monocytogenes infection in MZM/MM-depleted mice. LCL-treated (•) or control ({circ}) C57BL/6 mice were infected i.v. with 103 CFU of L. monocytogenes at day 0. Bacterial titers in spleen, liver, kidney, and lung were followed over time. Shown are individual values and means for four mice per group. Dashed lines represent detection limit of the assay. One representative experiment of three is shown. *, p < 0.05

 
APC function for the induction of specific immunity is not abrogated after selective depletion of MZM and MM

To further determine whether acquired immunity was induced in MZM/MM-depleted animals, development of Listeria-specific T cells was investigated in an adoptive transfer system. Control or LCL-treated C57BL/6-Thy-1.1 mice were primed with low dose listeriae to establish T cell immunity. After 25–30 days, splenocytes from mice of the two groups were transferred into sex-matched, nonirradiated wild-type C57BL/6-Thy-1.2 mice. Recipient mice were infected with listeriae, and expansion of donor-derived Listeria-specific T cells was followed by FACS analysis using a Thy-1.1-specific mAb. Day 1 postinfection, ~1.5–2.5% of CD8+ splenocytes in the peripheral blood of recipient mice were of donor origin. As shown in Fig. 7, donor-derived Listeria-specific CD8+ T cells expand from day 4 until day 15 in recipient mice, irrespective of whether or not donor mice were depleted of MZM/MM during primary infection.



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  		FIGURE 7. Listeria-specific T cells are induced in MZM/MM-depleted mice. LCL-treated or control C57BL/6-Thy-1.1 mice were infected i.v. with 103 CFU of L. monocytogenes; 25–30 days (d) after primary infection, 4 x 107 splenocytes from donor mice were transferred into sex-matched, nonirradiated wild-type C57BL/6-Thy-1.2 mice. Recipients were infected i.v. with 5 x 103 CFU of listeriae, and individual blood samples were examined for the expansion of donor-derived, Listeria-specific CD8+ T cells using FACS analysis. Percent Thy-1.1+CD8+ T cells of total CD8+ T cells is indicated. Shown is one representative experiment of three.

 
To further illustrate the finding that depletion of MZM/MM does not abrogate APC function and the induction of specific immunity, IFN-{gamma} levels and Listeria-specific Abs were determined in the serum during primary infection with listeriae (Fig. 8). IFN-{gamma} levels in the sera of Listeria-infected mice were detectable only during the first 7 days, with slightly elevated levels by day 3 in LCL-treated mice compared with control mice. The Listeria-specific Ab response in general was rather weak, and no differences between the MZM/MM-depleted and control animals could be demonstrated. These results indicate that in the absence of MZM/MM, the APC function in the spleen is not abrogated and Listeria-specific immunity is still induced.



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  		FIGURE 8. IFN-{gamma} production and induction of specific Ab response in MZM/MM-depleted mice is not impaired. LCL-treated ({blacksquare}) or control ({square}) C57BL/6 mice were infected i.v. with 103 CFU of L. monocytogenes and IFN-{gamma} (A) and Listeria-specific Abs (B) in the sera were determined at the indicated time points by ELISA. Results from three mice per group are given. Shown is one experiment of two.

 
To analyze whether induction of protective immunity was compromised by the depletion of MZM/MM, development of Listeria-specific T cell memory was investigated. Control or LCL-treated mice were primed with listeriae, and the memory T cell response was examined 30 days later. The two groups of primed mice and naive controls were challenged with 105 CFU of listeriae, and bacterial titers were determined in spleen and liver 1 and 2 days postinfection (Fig. 9). Irrespective of the presence or absence of MZM/MM during primary Listeria infection, mice were able to control challenge infection equally well, as indicated by the low bacterial load in spleen and liver. In contrast, unprimed mice failed to restrict listerial growth in the first 2 days after infection. Hence, absence of MZM/MM does not abrogate the induction of a protective Listeria-specific T cell response, demonstrating that Ag trapping and Ag presentation functions are not necessarily linked to the same cell type.



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  		FIGURE 9. Induction of protective, Listeria-specific T cell responses in MZM/MM-depleted mice. LCL-treated ({blacksquare}) or control ({square}) C57BL/6 mice were infected i.v. with 103 CFU of L. monocytogenes. Thirty days after primary infection, the two groups of memory mice as well as naive mice () were challenged with 105 CFU of L. monocytogenes. Bacterial titers in spleen and liver were determined at day (d) 1 and 2 after infection. Shown are mean values and SEM of three mice per group. Dashed lines represent detection limit of the assay. One representative experiment of two is shown. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we define the role of MZM/MM in early Ag trapping and in the immunological control of bacterial infection. Blood-borne particles like microspheres and L. monocytogenes were trapped almost exclusively in the splenic marginal zone promptly after i.v. administration. Weak fluorescence signals were detected in the red pulp, probably due to direct transport of Ag via small branches of the central artery ending in the red pulp directly, thereby bypassing the marginal zone (1, 2, 3). No Ag was detected in the white pulp. Our results support and extend findings of earlier localization studies using various particulate model Ags, like Indian ink, polystyrene nanoparticles, latex beads, liposomes differing in size and surface characteristics (6, 13, 14, 15, 16, 17), or soluble Ags like trinitrophenyl-Ficoll, hydroxyethyl starch, dextran, LPS, and immune complexes (5, 8, 9, 10, 11, 12). These Ags were trapped mainly in the marginal zone of the murine spleen after i.v. injection. Similar results were obtained when the initial clearing of circulating live pathogens was analyzed. Candida albicans, Brucella abortus, Lactobacillus acidophilus, L. monocytogenes, or vesicular stomatitis virus were trapped in the marginal zone early after application (18, 19, 20, 33). Although in some of these studies Ag uptake was demonstrated by macrophages of the marginal zone on the histological level, the trapping and clearing functions of the marginal zone was not functionally linked to distinct cell populations or correlated with control of infection.

In the present study, MZM and MM were selectively depleted by LCL treatment. In the absence of MZM/MM, trapping of particulate Ag in the marginal zone was lost, and microspheres and listeriae were scattered into the red pulp after i.v. injection. Interestingly, some marginal zone localization of Ag still exists, which may be explained by the particular anatomical organization of the marginal zone with a reduced blood flow due to open sinuses and the phagocytic activity of other cell populations. LCL administration did not deplete other cells of the marginal zone including marginal zone B cells, DC or MAdCAM-1-expressing reticular cells (34, 35). However, these cells could not compensate for the lack of MZM/MM and failed to maintain the original trapping capacity of the marginal zone. Thus, we prove that MZM/MM are crucial to trap particulate Ag in the marginal zone.

As a consequence of the impaired Ag trapping, MZM/MM-depleted mice experienced elevated hematogenic spread of listeriae to peripheral organs at early time points after high dose infection. The capacity of the liver to control listeriae during the first 6 h of infection, indicated by a characteristic 1-log reduction of bacterial burden, was not affected by depletion of MZM/MM. This is consistent with our observation that Kupffer cells are not eliminated by LCL treatment and sustain their effector functions. Even after low dose infection with listeriae, bacterial titers were significantly increased in all organs of MZM/MM-depleted animals during the first 4 days. In line with this observation, mice were more susceptible to sublethal Listeria infection. We conclude that an impaired Ag trapping in the marginal zone enhanced hematogenic spread of listeriae to peripheral organs, thereby reducing natural resistance. Earlier studies revealed that MZM/MM-depleted mice fail to control low dose infection with lymphocytic choriomeningitis virus, most probably due to spread of virus to peripheral organs during the early phase of infection followed by unrestricted virus replication (22). However, a functional link between early hematogenic spread of virus and impaired retention capacity of the marginal zone was not possible.

Complete depletion of resident splenic macrophages (MZM, MM, and red pulp macrophages) by high doses of clodronate liposomes also affected B cells and DC in the marginal zone and depleted Kupffer cells in the liver. This treatment renders mice more susceptible to sublethal doses of listeriae in primary infection and impairs acquired resistance against secondary Listeria infection (36, 37). Complete depletion of splenic macrophages eliminates also other cells in spleen and liver that are operative during early stages of primary and secondary Listeria infection; therefore, this regimen is not comparable with the selective depletion of a defined macrophage subset in the marginal zone of the spleen as described in this study.

Macrophages of the marginal zone not only express Ag trapping functions but also direct bacteriocidal effector functions. Discrimination of the two properties is impossible with the present experimental setup because depletion of MZM and MM abrogates both functions. Injection of UV-inactivated herpes simplex virus or vesicular stomatitis virus i.v. elevated production of IFN-{alpha}{beta} in the marginal zone promptly after infection (38, 39, 40). We consider it unlikely that IFN-{alpha}{beta} contributes directly to control of listeriosis because IFN-{alpha}{beta}-deficient mice can control this pathogen (41).

How can MZM and MM fulfill their unique trapping function? First, in the marginal zone, the terminal arterioles open into sinuses, and the blood flow is reduced. MZM and MM are located at this strategic position to trap and clear particulate and soluble Ags. Second, the macrophages of the marginal zone exhibit specific and exclusive receptor systems that may promote efficient Ag uptake. This is supported by the finding that particulate Ags (nanoparticles, fluoresceinated polysaccharides, Candida albicans) incubated in vitro on slices of living spleen tissues are mainly captured in the marginal zone (42, 43, 44), a localization pattern identical with that found in vivo. Therefore, in vivo trapping by the splenic marginal zone macrophages is most likely due to both their strategic anatomical location along the sinuses and their specific receptor repertoire.

Receptor-mediated recognition and phagocytosis are key events in host defense against pathogens (45, 46, 47). Opsonin-dependent phagocytosis via natural Abs seems to be important in early resistance against pathogens, by virtue of either their neutralizing capacity or opsonization for recruitment of Ag to secondary lymphoid organs. This is supported by the finding that mice lacking natural Abs exhibit elevated listerial titers in peripheral organs after high dose infection (48), demonstrating enhanced hematogenic spread, as observed in our study after depletion of MZM/MM. Components of the complement system are also critically involved in the control of bacterial and viral infections (49, 50, 51). MZM express CR3 abundantly, whereas red pulp macrophages are devoid of CR3. Therefore, direct binding of C3 breakdown products to a pathogen allows its trapping to MZM (48, 52).

Opsonin-independent phagocytosis is mediated by a variety of receptors including type I and II scavenger receptors, macrophage receptor with collagenous structure, CD36, the mannose receptor, and others (47, 53). Some of these receptors are abundantly expressed on MZM of the spleen (17, 54, 55). Mice deficient in type I and II scavenger receptors are more susceptible to L. monocytogenes and Staphylococcus aureus infection due to impaired capacity of macrophages to phagocytose and kill bacteria (56, 57). Taken together, natural Abs, components of the complement system, and expression of pattern recognition receptors contribute to the prominent Ag-trapping capacity of the macrophages in the marginal zone.

Several mutant mouse strains are defective in the microarchitecture of the marginal zone. Alymphoplastic (aly/aly) and osteopetrotic (op/op) mice or mouse strains with targeted disruptions in TNF, TNFR-1, lymphotoxin-{alpha}, lymphotoxin-{beta}, Pyk-2, RelB, NF-{kappa}B, or Bcl-3 gene loci show histological abnormalities in the marginal zone. MZM, MM, marginal zone B cells, and/or reticular endothelial cells are either disorganized or absent in these mice (58, 59, 60, 61, 62, 63, 64, 65, 66). Whether reduced Ag trapping in the marginal zone has an impact on the susceptibility of these animals to bacterial and viral infections is difficult to judge at present. In most of the cases, multiple factors including structural defects (lack of lymph nodes, follicular dendritic cell network, or germinal center formation) as well as impaired effector functions of immunocompetent cells or pathological alterations contribute to the abnormal immune system of such mice (67). The LCL protocol avoids such obstacles because with this technique MZM/MM were selectively eliminated without other cell populations being affected.

The marginal zone is not only an important site for early Ag trapping and clearance but may also represent a crucial compartment for Ag presentation and induction of specific immune responses. Complete depletion of MZM/MM which lasted for ~6–8 days had no adverse effects on the induction of Listeria-specific immunity. This was confirmed by the expansion of Listeria-specific T cells in an adoptive transfer system, the release of IFN-{gamma}, the production of Listeria-specific Abs, and the demonstration of protective immunity. Animals controlled infection by day 7 and eradicated bacteria by day 12, indicating that Ag presentation was not abrogated and protective Listeria-specific T cell responses were induced in the time frame when MZM/MM were absent. This implies that Ag trapping and Ag presentation function are not necessarily linked to the same cell type in the spleen. Other studies have shown that total depletion of splenic macrophages or block of MZM phagocytosis by Abs does not influence the humoral immune response against T-independent (TI) type II Ags (1, 11, 68). This has been anticipated because TI type II Ags activate B cells in the marginal zone directly without requiring Ag processing by MZM/MM (69), which allows a very early IgM-producing plasmablast response without involvement of T cells (70, 71, 72). However complete depletion of splenic macrophages probably also impaired DC function and subsequent Ag-specific T cell responses (73, 74). In contrast, the selective depletion of MZM and MM used in our study did not impair the induction of protective, Listeria-specific T cell responses.

In summary, our results emphasize the critical role of the marginal zone in early trapping of particulate Ag and identify the MZM and MM as the responsible cell population for this process. As a consequence, in the absence of MZM and MM, early control of Listeria infection was impaired due to enhanced hematogenic spread of the pathogen to peripheral organs. However, MZM and MM are dispensable for the induction of specific T cell responses and hence do not possess prominent Ag presentation function. This demonstrates that the Ag-trapping function and the Ag presentation function are not linked to the same cell type.


   Acknowledgments
 
We thank S. Bandermann and M. Stäber for excellent technical assistance and Drs. H. Collins, A. Sponaas, and U. Steinhoff for helpful discussions.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Stefan Kaufmann, Max-Planck-Institut für Infektionsbiologie, Abteilung Immunologie, Schumannstrasse 21, 10117 Berlin, Germany. E-mail address: kaufmann{at}mpiib-berlin.mpg.de Back

2 P.S. is supported by the Alexander von Humboldt-Stiftung. Back

3 Abbreviations used in this paper: DC, dendritic cells; MM, marginal metallophilic macrophages; MZM, marginal zone macrophages; LCL, low dose clodronate liposomes; MAdCAM-1, mucosal addressin cell adhesion molecule-1; TI, T cell independent. Back

Received for publication August 8, 2002. Accepted for publication May 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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