The JI Acurri Cytometers
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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ato, M.
Right arrow Articles by Kaye, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ato, M.
Right arrow Articles by Kaye, P. M.
The Journal of Immunology, 2004, 173: 4815-4820.
Copyright © 2004 by The American Association of Immunologists

Localization of Marginal Zone Macrophages Is Regulated by C-C Chemokine Ligands 21/191

Manabu Ato*, Hideki Nakano2,{dagger}, Terutaka Kakiuchi{dagger} and Paul M. Kaye3,*

* Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom; and {dagger} Department of Immunology, Toho University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The marginal zone (MZ) of the spleen is an important site for the capture of blood-borne pathogens and a gateway for lymphocytes entering the white pulp. We have recently reported that Leishmania donovani infection results in a remarkably selective loss of MZ macrophages (MZM) from the MZ. To understand the basis of this observation, we have investigated how MZM maintain their anatomical distribution in the steady state in uninfected mice. We now report that plt/plt mice, which lack functional CCL19 and CCL21, have significantly reduced numbers of MZM compared with normal C57BL/6 (B6) mice. Similarly, in B6.CD45.1->plt/plt chimeras, donor-derived MZM were rare compared with the number observed in reciprocal plt/plt->B6.CD45.1 chimeras. Moreover, we show that administration of pertussis toxin, an inhibitor of chemokine receptor signaling, to B6 mice results in exit of MZM from the MZ, that MZM can migrate in response to CCL19 and CCL21 in vitro, and that MZM colocalize with CD31+CCL21+ endothelial cells. Collectively, these data indicate that CCL21 and, to a lesser extent, CCL19 play significant roles in the distinctive localization of MZM within the splenic MZ. Deficiency of CCL19 and CCL21, as also previously observed in mice infected with L. donovani, may thus account for the selective loss of MZM seen during this infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The marginal zone (MZ)4 of the spleen is the boundary between the red pulp and the white pulp (1). It is comprised of unique MZ B cells (2), dendritic cells (3), and two types of macrophages enmeshed within a reticular cell network (4, 5). Marginal metallophilic macrophages (MMM) localize at the inner rim between the MZ and lymphocyte-rich white pulp, whereas MZ macrophages (MZM) are localized at the outer boundary of the MZ, adjacent to the red pulp. The MZ has various functions, including the capture of blood-borne pathogens (6), the regulation of lymphocyte trafficking into the white pulp (7), and the generation of Ag-specific B cell responses to T cell-independent Ags (8). Specifically, MZM are important for clearance of Leishmania donovani (9), lymphocytic choriomeningitis virus (10), and Listeria monocytogenesis (11). In some, but not all, cases, mice lacking MZM have been shown to be significantly more susceptible to infection and have altered T cell immune responses (11, 12).

Several studies using gene-targeted mice have shown that the development of the MZ is regulated through complex signaling pathways involving TNF superfamily members and transcription factors, such as those of the NF-{kappa}B family (reviewed in Ref. 13). Thus, lymphotoxin-{alpha} (LT-{alpha})-, NF-{kappa}B-inducing kinase-, or RelB-deficient mice completely lack a MZ (14, 15, 16), whereas NF-{kappa} B2-deficient mice have no MMM, but retain almost normal levels of MZM (17). Many of the cytokines and transcriptional factors regulating splenic architecture function by regulating chemokine expression (18, 19). Specific blockage of TNF superfamily members in normal adult mice has also demonstrated that these cytokines may play a role in the maintenance of lymphoid structure (20). Whether chemokines are essential for the steady state maintenance of MZM in their characteristic position in the MZ has not been determined, however.

We have recently been studying the dramatic remodeling of splenic microarchitecture that occurs as a result of chronic infection with L. donovani. Our studies indicate that two major structural changes occur: loss of MZM (21) and loss of gp38+ stromal cells from the T cell zone (22). Both events are mediated directly or indirectly by TNF. Given that gp38+ stromal cells are a major source of both CCL19 and CCL21, these findings lead us to postulate that the distribution of MZM in the spleen of normal uninfected mice might be regulated by chemotactic responses to these chemokines.

Mice bearing the paucity of lymph node T cells (plt) mutation (23) have a spontaneous recessive mutation in chromosome 4 that results in loss of both functional CCL19 and CCL21b genes (24). In this study we report that 1) MZM are selectively deficient in the MZ of plt/plt mice; 2) treatment of normal mice with pertussis toxin (PTx) results in the migration of MZM out of the MZ; 3) MZM can migrate in response to CCL19 and CCL21 in vitro; and 4) MZM colocalize with CCL21-expressing endothelial cells in the MZ. Together these findings lead us to conclude that the distribution of MZM is regulated by CCL21 and, perhaps to a lesser extent, CCL19 and propose that loss or aberrant expression of chemokines may underpin the extensive remodeling of lymphoid tissue noted in leishmaniasis and other chronic infections.


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

plt/plt mice backcrossed to the B6 background were provided by Drs. H. Hengartner and T. Junt (University of Zurich, Zurich, Switzerland). B6.CD45.1 mice were bred at the London School of Hygiene and Tropical Medicine under barrier conditions. B6 (CD45.2) mice were purchased from Charles River Laboratories (Margate, U.K.) and housed under specific pathogen-free conditions. Five hundred nanograms of PTx or PTx B oligomer (List Biological Laboratories, Campbell, CA) in saline was administrated by i.p. injection as indicated. For T cell depletion experiments, 500 and 250 µg of anti-CD4 (YTS 191.1) and anti-CD8 (YTS 169.4) mAb or control rat IgG (MAC5) was administrated by i.p. injection at 4 days and 1 day before killing, respectively.

Immunohistochemistry

Immunohistochemistry was performed on 6-µm frozen sections as described previously (9). Primary Abs were purified or biotinylated HL3 (anti-mouse CD11c), II/41 (anti-IgM), and A20 (anti-CD45.1; BD Pharmingen, San Jose, CA); 3D6.112 (anti-CD169), CI:A3–1(anti-F4/80), MCEA-367 (anti-MAdCAM-1), 390 (anti-CD31), KAT-1 (anti-ICAM-1; CD54), and M/K-2 (anti-VCAM-1; CD106; Serotec, Oxford, U.K.); ER-TR7 (BMA Biomedicals, Augst, Switzerland); anti-mouse CCL21 (R&D Systems, Abingdon, U.K.); ER-TR9 (anti-specific ICAM-3-grabbing nonintegrin-related gene 1; SIGNR1; a gift from G. Kraal, Free University, Amsterdam, The Netherlands); and FITC-conjugated anti-mouse IgD (11-26c.2a: BD Pharmingen). Secondary Abs were biotinylated rabbit anti-rat IgG (Vector Laboratories, Petersborough, U.K.), biotinylated rabbit anti-rat Ig (for ER-TR9; DakoCytomation, Ely, U.K.), donkey anti-goat IgG (for CCL21; Jackson ImmunoResearch Laboratories, West Grove, PA), Alexa 488-conjugated goat anti-rat IgG, or Alexa 546-conjugated streptavidin (Molecular Probes, Leiden, The Netherlands). As appropriate, sections were developed with Vector Elite-ABC kit followed by Vector 3,3'-diaminobenzidine substrate kit (Vector Laboratories), or directly viewed using a Zeiss LSM510 confocal microscope. In some experiments mice were injected i.v. with 200 µl of 5% (v/v, in 0.9% NaCl) India ink (Rowney and Company, Brackwell, U.K.) to identify MZM.

Flow cytometry

MZ B cells were enumerated by flow cytometry using RA3-6B2 (anti-CD45R/B220) and KMC8 (anti-CD9). To evaluate MZM, 100 µg of FITC-conjugated dextran (Molecular Probes) was administrated i.v. to B6 mice. After 45 min, spleens were harvested and digested in RPMI 1640 (Invitrogen Life Technologies, Paisley, U.K.) containing 0.1% collagenase D (Roche, Mannheim, Germany) and 100 µg/ml DNase I (Sigma-Aldrich, Dorset, U.K.) at 37°C for 30 min. After washing with calcium-free medium, cells were resuspended with 2 ml of Ca-free PBS containing 5 mM EDTA and 1% FCS (Sigma-Aldrich) and were incubated with magnetic microbeads conjugated with anti-mouse CD19 (1D3) and anti-mouse Thy1.2 (30-H12; Miltenyi Biotec, Bergisch Gladbach, Germany), then B and T cells were negatively removed by magnetic sorting (Miltenyi Biotec). Cells were stained for flow cytometry to identify ER-TR9+ FITC+ MZM or elucidate CCR7 expression with CCL19-Ig (a gift from Drs. T. Springer and U. von Andrian, Harvard Medical School, Boston, MA) (25) and were analyzed using FACSCalibur (BD Biosciences, Mountain View, CA).

Bone marrow (BM) transplantation

BM chimeric mice were prepared as previously described (26). Briefly, recipient mice were irradiated twice (48 h apart) with 5.5 Gy, then engrafted with 107 donor-derived BM cells i.v. via the tail vein. After 4 wk, PBLs in chimeric mice were >98% donor derived (data not shown). Mice were injected i.v. with 200 µl of 5% India ink 24 h before killing.

In vitro migration assay

Migration assays were conducted as previously described (22) with modification. Briefly, assays were performed in Transwell inserts (5-µm pore size; Fisher Scientific, Longborough, U.K.) placed in 24-well plates containing 500 µl of medium, CCL19, or CCL21 solution (R&D Systems). After 3-h incubation, cells in the lower wells were collected, and 104 10-µm microsphere beads were added (Polysciences, Warrington, PA). Cells were stained for flow cytometry to identify ER-TR9+ FITC+ MZM and were analyzed using FACSCalibur (BD Biosciences). The migration index is calculated as the number of MZM migrating in response to a chemokine divided by the number migrating in medium alone.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MZM are selectively deficient in plt/plt mice

In mice chronically infected with L. donovani, we previously observed that the expression of CCL19 and CCL21 by stromal cells was dramatically reduced (22) and that MZM were selectively lost from their normal position at the outer edge of the MZ (21). To determine whether these two events might be causally linked, we assessed the importance of CCL19 and CCL21 in the regulation of MZM positioning within the MZ of normal and chemokine-deficient (plt/plt) mice (Fig. 1). In normal B6 mice, a discrete population of ER-TR9+ MZM capable of taking up injected India ink was clearly evident (Fig. 1A). In contrast, MZM as defined by either ER-TR9 staining or India ink uptake were significantly reduced in number in plt/plt mice (Fig. 1B). CD169+ MMM, which localized at the inner rim of the MZ in B6 mice (Fig. 1C), were also noted to be distributed in a broader band in plt/plt mice and extended into the white pulp (Fig. 1D). The distribution of F4/80+ red pulp macrophages was not significantly different when comparing B6 and plt/plt mice (Fig. 1, E and F). plt/plt mice had no gross defects in the positioning of various endothelial populations, as defined by staining with CD54 and CD106 (data not shown). Furthermore, variation in T cell numbers in plt/plt mice compared with B6 mice was unlikely to be of importance, because depletion of T cells in B6 mice did not influence the number or localization of MZM (data not shown). To further quantify the loss of MZM in plt/plt mice we used two approaches. First, the number of MZM was directly counted in tissue sections, on the basis of India ink uptake (Fig. 1G). Second, the frequency of MZM was determined in total spleen cell populations by staining with ER-TR9 (Fig. 1H). Both approaches confirmed the deficiency of MZM in plt/plt mice compared with B6 mice.



View larger version (80K):
[in this window]
[in a new window]
  		FIGURE 1. plt/plt mice have reduced numbers of MZM compared with B6 mice. India ink was injected the day before mice were killed. Six-micron frozen sections were cut from the spleens from B6 (A, C, and E), or plt/plt (B, D, and F) mice. A and B, India ink and ER-TR9 were used to detect MZM; C and D, India ink and CD169 were used for MZM and MMM, respectively; E and F, F4/80 for red pulp macrophages. Original magnification, x100. G, The number of India ink-positive MZM was determined from ~10 fields/mouse (n = 4–6 mice) in B6 vs plt/plt mice. H, The frequency of ER-TR9+ MZM was determined in B6 and plt/plt mice by flow cytometry. Data are representative of one of five experiments performed. *, p < 0.05.

 
Radioresistant cells in plt/plt mice are responsible for the deficiency of MZM

To exclude the possibility that the chromosomal deletion found in plt/plt mice disrupted genes essential for the development of MZM, we established reciprocal BM chimeras between B6.CD45.1 and plt/plt (CD45.2) mice. As anticipated (23), both plt/plt mice and B6.CD45.1->plt/plt mice had poorly developed T cell areas compared with either plt/plt->B6.CD45.1 or B6->B6.CD45.1 mice (data not shown). To unambiguously identify MZM as being of donor or recipient origin, we used a combination of India ink uptake and CD45.1 staining. The plt/plt->B6.CD45.1 and B6->B6.CD45.1 mice had almost normal numbers of MZM, as visualized by India ink uptake or ER-TR9 Ab (Fig. 2A and data not shown). Staining for CD45.1 identified a small number of residual recipient-derived cells in these chimeras, but ~90% of the cells that took up India ink were clearly negative for CD45.1 expression, indicating their donor origin (Fig. 2, C and D). However, the total number of MZM in B6.CD45.1->plt/plt mice was significantly reduced (Fig. 2, B and E) to the level seen in plt/plt mice (c.f., Fig. 1, A and G). In these BM chimeras, the distribution of MMM showed either narrow or broad distribution in keeping with the genotype of the recipient (Fig. 2, A and B). Thus, the lack of MZM in plt/plt mice is not due to a defect in the capacity of plt/plt BM progenitors to develop into MZM.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 2. MZM can be derived from plt/plt bone marrow precursors. India ink was injected the day before mice were killed. Six-micron frozen sections were cut from the spleens from plt/plt->B6.CD45.1 (A) or B6.CD45.1->plt/plt (B) mice. India ink and CD169 were used for MZM and MMM, respectively. Original magnification, x100. C and D, CD45.1 staining was performed on spleen sections from plt/plt->B6.CD45.1 (C) or B6->B6.CD45.1 (D) mice. Original magnification, x400. E, The number of India ink-positive MZM was determined from ~10 fields/mouse (n = 4 mice) in B6 vs plt/plt mice. *, p < 0.05. n.s., not significant.

 
plt/plt mice have normal levels of MZ B cells

Recently, it was reported that interactions between MZM and MZ B cells are necessary to maintain MZ structure (27). To rule out the possibility that changes to MZ B cells in plt/plt mice might have occurred with reciprocal effects on MZM distribution, we stained splenocytes from plt/plt and B6 mice with B220 and CD9 (Fig. 3A), a new marker of MZ B cells that is not expressed on spleen follicular B cells (28). No significant difference was seen in either the proportion (Fig. 3B) or total number (Fig. 3C) of MZ B cells in these mice. Furthermore, the distribution of MZ B cells (identified by IgM and IgD staining) in the spleen of B6 and plt/plt mice was identical (Fig. 2D). This result is consistent with data from a recent study that indicated MZ B cell numbers and distribution are normal in CCR7/CXCR5 double-deficient mice (29). Taken together, our data suggest that the absence of MZM from the spleen in plt/plt mice is not attributable to defective interaction between MZM and MZ B cells, but, rather, is a direct consequence of the lack of CCL19 and/or CCL21.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 3. plt/plt mice have normal numbers of MZ B cells. Splenocytes from plt/plt mice and B6 mice were stained for B220 and CD9. A, The flow cytometry profile is representative of at least three mice, and the numbers indicate the percentage of MZ B cells. B and C, MZ B cells given as a percentage of total B cells (B) and in terms of absolute number (C) in plt/plt mice and B6 mice. Data show the mean ± SE from at least three mice of each strain. D, Histological distribution of MZ B cells in plt/plt mice and B6 mice. Six-micron frozen spleen sections were stained for IgD (green) and IgM (red). Original magnification, x200.

 
PTx treatment induces redistribution of MZM

To formally demonstrate that active chemokine secretion is responsible for maintenance of MZM in their distinctive position within the MZ, we blocked chemokine receptor signaling in vivo by the administration of PTx, an inhibitor of GTP-binding proteins including chemokine receptors (30). After 3 days of PTx treatment, India ink-positive MZM had begun to exit the MZ and were found within the red pulp (Fig. 4). India ink-positive cells were confirmed as MZM by ER-TR9 staining, a result that also served to demonstrate that India ink had not been released after PTx treatment and then phagocytosed by red pulp macrophages (data not shown). After 6 days of PTx treatment, MZM were rarely seen in the MZ, but some could still be observed throughout the red pulp. In contrast, mice treated with PTx B oligomer did not alter the distribution of MZM (Fig. 4). The distribution of F4/80+ red pulp macrophages was not significantly altered, except for some compression due to the increased granulocyte numbers also associated with PTx treatment (data not shown). These results strongly support that idea that continuous chemokine signaling is necessary for the maintenance of position of MZM in the murine spleen.



View larger version (185K):
[in this window]
[in a new window]
  		FIGURE 4. PTx treatment leads to exit of MZM from the MZ. India ink was injected the day before PTx treatment. India ink-positive MZM and CD169+ MMM were shown in 6-µm frozen sections from the spleen of control PTx B oligomer-treated (A, C, and E), or PTx-treated (B, D, and F) mice at 1 day (A and B), 3 days (C and D), or 6 days (E and F) after treatment. Data are representative of one of three experiments performed. Original magnification, x200.

 
MZM migrate in response to CCL19 and CCL21

The studies described above using mutant mice and an in vivo antagonist of chemokine receptor signaling all suggest that MZM are responsive to the chemotactic activity of CCL19 and/or CCL21. To directly confirm whether MZM can indeed migrate in response to these specific chemokines, we enriched these cells and performed in vitro migration assays. To aid identification of MZM in vitro, we injected mice with FITC-dextran, a ligand for mSIGNR1 expressed on MZM (31, 32). MZ B cells, which may also bind FITC dextran (32), were removed by magnetic sorting from spleen cell suspensions before assay. Within the resultant spleen cell populations, FITC+CD11c–B220– cells were homogeneously positive for ER-TR9, and we regarded them as MZM (Fig. 5A). Staining with a CCL19-Ig fusion protein (25) indicated that these cells weakly expressed CCR7, at a level below that seen on naive T cells or CD11chigh dendritic cells (Fig. 5B). In vitro, CCL21 stimulated migration of MZM in a dose-dependent fashion, with an optimal response at 100 nM. Similarly, migration was observed to CCL19, with an optimal response at 200 nM. The absolute level of migration to CCL19 at the doses tested was nevertheless consistently lower than that seen with CCL21 (Fig. 5C). Thus, we have demonstrated for the first time that MZM have the capacity to respond directly to these two constitutively expressed chemokines.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. MZM migrate in response to CCL19 and CCL21 in vitro. FITC-dextran was injected into normal B6 mice, and spleens were removed 45 min later. Chemotactic responses of B and T cell-depleted spleen cells were assessed using a Transwell system. A and B, FITC+CD11c–B220– cells were stained with A) ER-TR9 (open histogram) or isotype control (closed histogram) or B) CCL19-Ig (open histogram) or control human Ig (closed histogram). C, The migration index of MZM in response to CCL19 ({blacktriangleup}) and CCL21 ({blacksquare}) is shown as the mean ± SE. These experiments were conducted at least twice with similar results (n = 3 mice/experiment). *, p < 0.05.

 
Expression of CCL21 on CD31+ endothelial cells, but not MAdCAM-1+ marginal sinus lining cells

The data shown above demonstrate that CCL21 can attract MZM in vitro. To evaluate whether in vivo expression of CCL21 is associated with distribution of MZM in the MZ, we stained spleen sections for CCL21 and stromal cell markers. As previously reported, CCL21 was mainly expressed by stromal cells in the T cell area of the spleen (19) as well as in the MZ area (Fig. 6). The specificity of CCL21 staining was confirmed by the lack of staining of spleen sections from plt/plt mice. No significant difference in the distribution of stromal cell markers MAdCAM-1, CD31, and ER-TR7 were observed between B6 and plt/plt mice (Fig. 6, B, D, and F). Double staining with CCL21 and MAdCAM-1 showed that CCL21 expression in the MZ was not associated with the MAdCAM-1+ marginal sinus, but, rather, with cells in the outer perimeter of the MZ (Fig. 6A). In contrast, CCL21 was found coexpressed on CD31+ endothelial cells in the outer MZ (Fig. 6C) and occasionally on ER-TR7+ stromal cells (Fig. 6E). In contrast, CCL19 expression, although readily observed in the periarteriolar T cell regions of the white pulp, was not detectable on cells within the MZ (22) (data not shown). Given the expression of CCL21 in the outer MZ, we lastly examined the distribution of MZM in relation to the position of CCL21+ cells. India ink-positive MZM were found in many instances to be in close proximity to CD31+ CCL21+ endothelial cells (Fig. 6G) Hence, these data indicate that MZM are positioned in the outer MZ coincident with expression of CCL21.



View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 6. Tissue expression of CCL21 in plt/plt mice and B6 mice. Six-micron frozen spleen sections from B6 (A, C, and E) and plt/plt (B, D, and F) mice were stained for MAdCAM-1 (A and B), CD31 (C and D), or ER-TR7 (E and F; green) and CCL21 (red). Original magnification, x200. Magnification for insets in C and E, x350. G, Spleen section from B6 mouse injected with India ink to show MZM (black) and stained for expression of CCL21 (red) and CD31 (green) with superimposed Nomarsky image. Arrows indicate CD31+ cells strongly expressing CCL21 (orange) adjacent to MZM. Magnification, x200. wp, white pulp.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The precise development of lymphoid tissue architecture plays an important role during ontology in establishing an appropriate environment for the efficient generation of immune responses and is tightly regulated by stimuli acting through TNF superfamily receptors (33). Notably, LT{alpha}1{beta}2-LT{beta}R interactions play an important role in orchestrating splenic architecture, including organization of the MZ and B cell follicles (14). Recently, a role for B cells in the development and maintenance of the MZ has been described (34). However, there have been few reports on the nature of the factor(s) responsible for directing and maintaining the positioning of discrete macrophage populations in the various spleen compartments. To our knowledge, this is the first report demonstrating that continuous chemokine signaling, in particular CCL21, is necessary for the localization of MZM at their unique position within the MZ.

A number of observations have lead to the conclusion that CCL21 and CCL19 play an important role in the localization of MZM to the MZ; MZM express CCR7 and migrate in response to CCL21 and CCL19 in vitro, MZM distribution in normal mice is reflected in the distribution of CCL21+ CD31+ endothelial cells in the outer MZ, and pharmacologic and genetic disruption of CCL21/CCL19 function interferes with MZM positioning. Furthermore, the absence of detectable CCL19 expression in the MZ suggests that CCL21 may play the most dominant role in MZM localization in vivo. Although our data clearly implicate these chemokines in MZM positioning, the retention of a few MZM in the MZ of plt/plt mice suggests that there may be some redundancy in the signals required. In addition, the common involvement of CCL19/CCL21 in the migration of MZM, T cells (35), and dendritic cells (36) raises several new questions concerning the anatomical segregation of these cells that occurs in the resting spleen. MZM express low levels of CCR7 and respond more weakly than either T cells or dendritic cells in in vitro migration assays. Hence, MZM migration into the periarteriolar region, where stromal cells express abundant CCL21 and CCL19 (22), may be more readily opposed by other chemokines and be more readily affected by altered levels of chemokine expression. Indeed, in our studies of dendritic cell migration, we clearly demonstrated that even the minimal level of CCL21 expressed on the endothelium of the central arterioles in L. donovani-infected mice was sufficient to recruit CCR7+CD11chigh dendritic cells from the MZ into the periarteriolar region (22). It is also possible that MZM positioning is influenced by the coordinate action of multiple other chemokines acting with CCL19 and/or CCL21, as, for example, in the case of CXCL12 (37). The MAdCAM-1+ endothelium of the marginal sinus has been suggested to have a role in retention of MMM in the MZ (38), but no differences in architecture of the marginal sinus were observed in plt/plt mice, suggesting that the structural elements of the MZ were indeed intact in these chemokine-deficient mice. However, CCL21 expression was observed on both the endothelial cells of the central arterioles as well as on CD31+ endothelial cells in the MZ of B6 mice, whereas it was absent in plt/plt mice. The observation that MZM are in close proximity to these CCL21+CD31+ cells would also be compatible with a model in which MZM additionally engage in stronger adhesive interactions with stromal elements found in MZ compared with T cells and dendritic cells. Additional studies are required to distinguish between these possibilities.

The results of this study now also suggest an explanation for our previous finding that MZM are lost from the spleen of mice with chronic L. donovani infection. We previously demonstrated that loss of MZM was a result of excess TNF production, in that it was less evident in TNF-deficient mice (21). Excess TNF was also associated with a striking loss of T zone stromal cells, an important source of CCL19 and CCL21 in the spleen. Hence, the expression of CCL19 and CCL21, as determined by immunohistochemistry, is greatly reduced in infected mice (22). Our new data suggest that these events are causally related, and that TNF-mediated loss of chemokine production is the mechanism underlying the emigration of MZM from the MZ during infection.

In conclusion, this study indicates that the localization of MZM is largely regulated through signals delivered by CCL21 and perhaps, to a lesser extent, CCL19, providing a novel mechanism for the loss of organization of macrophages as seen in murine leishmaniasis. The importance of these or other chemokines for the maintenance of macrophage positioning in human disease remains to be determined.


   Acknowledgments
 
We thank the staff of the Biological Services Facility for assistance with the breeding and maintenance of mouse colonies, Drs. Hengartner and Junt for initially providing B6-plt/plt mice, and Dr. C. Engwerda for critical comments on the manuscript.


   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 by the Wellcome Trust and the British Medical Research Council. M.A. is a Wellcome Trust International Traveling Fellow. Back

2 Current address: Department of Immunology, Duke University Medical Center, Durham, NC 27710. Back

3 Address correspondence and reprint requests to Dr. Paul M. Kaye, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, U.K. WC1E 7HT. E-mail address: paul.kaye{at}lshtm.ac.uk Back

4 Abbreviations used in this paper: MZ, marginal zone; BM, bone marrow; LT, lymphotoxin; MZM, MZ macrophage; MMM, marginal metallophilic macrophage; PTx, pertussis toxin. Back

Received for publication April 5, 2004. Accepted for publication August 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kraal, G.. 1992. Cells in the marginal zone of the spleen. Int. Rev. Cytol. 132:31.[Medline]
  2. Martin, F., J. F. Kearney. 2002. Marginal zone B cells. Nat. Rev. Immunol. 2:323.[Medline]
  3. Leenen, P. J., K. Radosevic, J. S. Voerman, B. Salomon, N. van Rooijen, D. Klatzmann, W. van Ewijk. 1998. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J. Immunol. 160:2166.[Abstract/Free Full Text]
  4. Kraal, G., M. Janse. 1986. Marginal metallophilic cells of the mouse spleen identified by a monoclonal antibody. Immunology 58:665.[Medline]
  5. 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.[Medline]
  6. Humphrey, J. H., D. Grennan. 1981. Different macrophage populations distinguished by means of fluorescent polysaccharides: recognition and properties of marginal-zone macrophages. Eur. J. Immunol. 11:221.[Medline]
  7. Lyons, A. B., C. R. Parish. 1995. Are murine marginal-zone macrophages the splenic white pulp analog of high endothelial venules?. Eur. J. Immunol. 25:3165.[Medline]
  8. Humphrey, J. H.. 1985. Splenic macrophages: antigen presenting cells for TI-2 antigens. Immunol. Lett. 11:149.[Medline]
  9. Gorak, P. M., C. R. Engwerda, P. M. Kaye. 1998. Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection. Eur. J. Immunol. 28:687.[Medline]
  10. Odermatt, B., M. Eppler, T. P. Leist, H. Hengartner, R. M. Zinkernagel. 1991. Virus-triggered acquired immunodeficiency by cytotoxic T-cell-dependent destruction of antigen-presenting cells and lymph follicle structure. Proc. Natl. Acad. Sci. USA 88:8252.[Abstract/Free Full Text]
  11. Aichele, P., J. Zinke, L. Grode, R. A. Schwendener, S. H. Kaufmann, P. Seiler. 2003. Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J. Immunol. 171:1148.[Abstract/Free Full Text]
  12. Seiler, P., P. Aichele, B. Odermatt, H. Hengartner, R. M. Zinkernagel, R. A. Schwendener. 1997. Crucial role of marginal zone macrophages and marginal zone metallophils in the clearance of lymphocytic choriomeningitis virus infection. Eur. J. Immunol. 27:2626.[Medline]
  13. Weih, F., J. Caamaño. 2003. Regulation of secondary lymphoid organ development by the nuclear factor-{kappa}B signal transduction pathway. Immunol. Rev. 195:91.[Medline]
  14. Fu, Y. X., D. D. Chaplin. 1999. Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17:399.[Medline]
  15. Koike, R., T. Nishimura, R. Yasumizu, H. Tanaka, Y. Hataba, Y. Hataba, T. Watanabe, S. Miyawaki, M. Miyasaka. 1996. The splenic marginal zone is absent in alymphoplastic aly mutant mice. Eur. J. Immunol. 26:669.[Medline]
  16. 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.[Abstract/Free Full Text]
  17. Franzoso, G., L. Carlson, L. Poljak, E. W. Shores, S. Epstein, A. Leonardi, A. Grinberg, T. Tran, T. Scharton-Kersten, M. Anver, et al 1998. Mice deficient in nuclear factor (NF)-{kappa}B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187:147.[Abstract/Free Full Text]
  18. Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, M. D. Cooper, J. L. Browning, J. D. Sedgwick, J. G. Cyster. 1999. Lymphotoxin {alpha}/{beta} and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189:403.[Abstract/Free Full Text]
  19. 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.[Medline]
  20. Mackay, F., G. R. Majeau, P. Lawton, P. S. Hochman, J. L. Browning. 1997. Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice. Eur. J. Immunol. 27:2033.[Medline]
  21. Engwerda, C. R., M. Ato, S. E. Cotterell, T. L. Mynott, A. Tschannerl, P. M. Gorak-Stolinska, P. M. Kaye. 2002. A role for tumor necrosis factor-{alpha} in remodeling the splenic marginal zone during Leishmania donovani infection. Am. J. Pathol. 161:429.[Abstract/Free Full Text]
  22. Ato, M., S. Stäger, C. R. Engwerda, P. M. Kaye. 2002. Defective CCR7 expression on dendritic cells contributes to the development of visceral leishmaniasis. Nat. Immunol. 3:1185.[Medline]
  23. Nakano, H., T. Tamura, T. Yoshimoto, H. Yagita, M. Miyasaka, E. C. Butcher, H. Nariuchi, T. Kakiuchi, A. Matsuzawa. 1997. Genetic defect in T lymphocyte-specific homing into peripheral lymph nodes. Eur. J. Immunol. 27:215.[Medline]
  24. Nakano, H., S. Mori, H. Yonekawa, H. Nariuchi, A. Matsuzawa, T. Kakiuchi. 1998. A novel mutant gene involved in T-lymphocyte-specific homing into peripheral lymphoid organs on mouse chromosome 4. Blood 91:2886.[Abstract/Free Full Text]
  25. Manjunath, N., P. Shankar, J. Wan, W. Weninger, M. A. Crowley, K. Hieshima, T. A. Springer, X. Fan, H. Shen, J. Lieberman, et al 2001. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108:871.[Medline]
  26. Engwerda, C. R., T. L. Mynott, S. Sawhney, J. B. De Souza, Q. D. Bickle, P. M. Kaye. 2002. Locally up-regulated lymphotoxin {alpha}, not systemic tumor necrosis factor {alpha}, is the principle mediator of murine cerebral malaria. J. Exp. Med. 195:1371.[Abstract/Free Full Text]
  27. 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.[Abstract/Free Full Text]
  28. Won, W. J., J. F. Kearney. 2002. CD9 is a unique marker for marginal zone B cells, B1 cells, and plasma cells in mice. J. Immunol. 168:5605.[Abstract/Free Full Text]
  29. Ohl, L., G. Henning, S. Krautwald, M. Lipp, S. Hardtke, G. Bernhardt, O. Pabst, R. Föster. 2003. Cooperating mechanisms of CXCR5 and CCR7 in development and organization of secondary lymphoid organs. J. Exp. Med. 197:1199.[Abstract/Free Full Text]
  30. Cyster, J. G., C. C. Goodnow. 1995. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J. Exp. Med. 182:581.[Abstract/Free Full Text]
  31. 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.[Abstract/Free Full Text]
  32. Kang, Y. S., S. Yamazaki, T. Iyoda, M. Pack, S. A. Bruening, J. Y. Kim, K. Takahara, K. Inaba, R. M. Steinman, C. G. Park. 2003. SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen, mediates uptake of the polysaccharide dextran. Int. Immunol. 15:177.[Abstract/Free Full Text]
  33. Gommerman, J. L., J. L. Browning. 2003. Lymphotoxin/light, lymphoid microenvironments and autoimmune disease. Nat. Rev. Immunol. 3:642.[Medline]
  34. 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.[Abstract/Free Full Text]
  35. Kellermann, S. A., S. Hudak, E. R. Oldham, Y. J. Liu, L. M. McEvoy. 1999. The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3{beta} are potent chemoattractants for in vitro- and in vivo-derived dendritic cells. J. Immunol. 162:3859.[Abstract/Free Full Text]
  36. Gunn, M. D., K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. Natl. Acad. Sci. USA 95:258.[Abstract/Free Full Text]
  37. Okada, T., V. N. Ngo, E. H. Ekland, R. Förster, M. Lipp, D. R. Littman, J. G. Cyster. 2002. Chemokine requirements for B cell entry to lymph nodes and Peyer’s patches. J. Exp. Med. 196:65.[Abstract/Free Full Text]
  38. Wang, C. C., C. Biben, L. Robb, F. Nassir, L. Barnett, N. O. Davidson, F. Koentgen, D. Tarlinton, R. P. Harvey. 2000. Homeodomain factor Nkx2–3 controls regional expression of leukocyte homing coreceptor MAdCAM-1 in specialized endothelial cells of the viscera. Dev. Biol. 224:152.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Bajenoff, N. Glaichenhaus, and R. N. Germain
Fibroblastic Reticular Cells Guide T Lymphocyte Entry into and Migration within the Splenic T Cell Zone
J. Immunol., September 15, 2008; 181(6): 3947 - 3954.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
M. A. Nolte and R. A.W. van Lier
The price of the CD27-CD70 costimulatory axis: you can't have it all
J. Exp. Med., October 30, 2006; 203(11): 2405 - 2408.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
M. Matter, B. Odermatt, H. Yagita, J.-M. Nuoffer, and A. F. Ochsenbein
Elimination of chronic viral infection by blocking CD27 signaling
J. Exp. Med., September 4, 2006; 203(9): 2145 - 2155.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Ato, A. Maroof, S. Zubairi, H. Nakano, T. Kakiuchi, and P. M. Kaye
Loss of Dendritic Cell Migration and Impaired Resistance to Leishmania donovani Infection in Mice Deficient in CCL19 and CCL21
J. Immunol., May 1, 2006; 176(9): 5486 - 5493.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Zaft, A. Sapoznikov, R. Krauthgamer, D. R. Littman, and S. Jung
CD11chigh Dendritic Cell Ablation Impairs Lymphopenia-Driven Proliferation of Naive and Memory CD8+ T Cells
J. Immunol., November 15, 2005; 175(10): 6428 - 6435.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. E. Berg, E. Crossley, S. Murray, and J. Forman
Relative Contributions of NK and CD8 T Cells to IFN-{gamma} Mediated Innate Immune Protection against Listeria monocytogenes
J. Immunol., August 1, 2005; 175(3): 1751 - 1757.
[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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ato, M.
Right arrow Articles by Kaye, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ato, M.
Right arrow Articles by Kaye, P. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS