Immunoarchitecture of Distinct Reticular Fibroblastic Domains in the White Pulp of Mouse Spleen
Departments of Immunology and Biotechnology (PB) and Radiotherapy and Oncology (GH), Faculty of Medicine, University of Pécs, Hungary, and Immunology Group (AKS), Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, Virginia
Correspondence to: Péter Balogh, Dept. of Immunology and Biotechnology, University of Pécs, Szigeti út 12, 7643 Pécs, Hungary. E-mail: peter.balogh{at}aok.pte.hu
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Summary |
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(J Histochem Cytochem 52:12871298, 2004)
Key Words: spleen white pulp stroma fibroblast heterogeneity SCID mouse
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Introduction |
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The spleen, as the single largest peripheral lymphoid organ in both humans and rodents, has a number of distinguishing features compared with other tissues of the immune system. The main parts of the spleen are the white pulp and the red pulp, with distinct and complementary functions. The boundary between the two regions is the marginal zone, containing specialized marginal sinus-lining cells surrounded by a fibroblastic reticular network, marginal zone macrophage subsets, and a separate B-cell pool (Kraal 1992; Martin and Kearney 2002
; Karlsson et al. 2003
). In the white pulp, the lymphocyte composition of distinct T- and B-cellrich compartments is similar to that in lymph nodes (Nolte et al. 2000
). In the white pulp, the T-cells encircle the central arteriole, thus forming the periarteriolar lymphoid sheath (PALS), to which spheroid accumulations of B-cells and follicular dendritic cells as follicles are connected. The red pulp may serve as an auxillary myelopoietic compartment and functions as an exit route for the majority of recirculating lymphoid cells. In addition, it efficiently phagocytoses blood-borne antigens and decaying erythrocytes via its macrophages, and hosts clusters of plasma cells (MacLennan et al. 2003
).
The movement of recirculating lymphoid cells and the flow of soluble compounds are greatly influenced by the mesenchymal scaffolding of the spleen, composed of reticular cells and a broad array of extracellular matrix components associated with these cells (Liakka and Autio-Harmainen 1992; Ocklind et al. 1993
). This guiding function may also include the adsorption and selective display of homing chemokines produced by regional (follicular or T-cell zoneassociated) stromal cells as directional cues (Nolte et al. 2003
). In this complex arrangement, the fibroblastic reticular cells create a meshwork that may connect distant parts of the splenic lymphoid tissue and may thus contribute to the formation of a continuous route for migrating cells and soluble molecules as well, similarly to lymph nodes (Gretz et al. 1996
). Furthermore, the possible role of reticular cells in influencing specific immune responses has been indicated by the trapping and possible retention of immune complexes coated via complement by ill-defined perivascular reticular cells (Taylor et al. 2002
). Moreover, the addition of fibroblast cells in culture could promote the formation of plasma cells after in vitro activation of human B-cells, in which system the extent of this supportive effect was dependent on the tissue origin of fibroblastic cells (Skibinski et al. 1998
).
Although many of the molecular mechanisms that contribute to the lymphocyte migration and positioning remain obscure, many aspects of the splenic fibroblastic reticular cells have remained enigmatic. A limited number of studies indicate their regional heterogeneity with regard to their phenotypic markers (Yoshida et al. 1991,1993
). However, data concerning their developmental and functional specifics are sparse (ten Dam et al. 2003
). In murine studies aimed at the splenic reticular architecture, the fibroblasts have been identified with the rat monoclonal antibody ER-TR7 (Van Vliet et al. 1986
) against an undefined marker that, although providing valuable information on the general fibroblastic arrangement of the spleen and other lymphoid organs, does not reveal the regional complexity of the reticular meshwork. Therefore, any new reagent that can identify subset-restricted elements of the reticular fibroblastic compartments may be of potential use in providing more accurate data on the dynamics and architecture of the lymphoid-mesenchymal domains of the spleen.
Here we report the production of two new rat monoclonal antibodies (MAbs) directed against different elements of the splenic fibroblast network. Using these antibodies, we also analyze the postnatal appearance of distinct fibroblast subsets and their spatial relationship in both normal and SCID mice, and also in chimeric SCID mice reconstituted with mature lymphoid cells. Our data indicate that the reticular scaffolding in various parts of the splenic white pulp is composed of phenotypically different fibroblasts whose physiological tissue arrangement in normal mice requires the presence of lymphocytes.
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Materials and Methods |
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Hybridoma Production
Splenic white pulp fragments from BALB/c mice that had been immunized IP with washed splenic red blood cells (SRBCs) were collected as described previously (Nolte et al. 2000). After isolation, the loose cells were separated from the fragments with repeated sedimentation at 1 x g in DMEM tissue culture medium, followed by injection into 8-week-old Wistar rats (white pulp material equivalent to five spleens on three occasions, 2 weeks apart each). The spleen cells from immunized Wistar rats were fused with Sp-2/0 Ag 14 myeloma-derived cells using polyethylene glycol MW 4000 and processed as previously described (Köhler and Milstein 1975
). The hybridoma supernatants were tested for specific antibody production using immunohistochemistry (Balázs et al. 1999
); the positive wells were cloned by repeated limiting dilution.
Antibodies and Other Reagents
Anti-endothelium and pan-reticular rat MAb IBL-7/22, DaB1 mouse alloantibody (IgG1 isotype) against MHC Class I (H-2Kb allotype), FITC-labeled rat anti-mouse CD45 (clone IBL-5/25), and anti-mouse MHC Class II (IBL-5/22) MAbs were produced in our lab (Balázs et al. 1998,1999
, 2001
). Antibodies against murine MadCAM-1 (clone MECA-89), VCAM-1 (clone 429), CR1/2: CD35/21 (clone 7G6), and B220/CD45RA (clone RA3-6B2) were purchased from BD Pharmingen (San Diego, CA). The anti-mouse CD3 MAb clone KT3 was obtained from Serotec (Crawley Down, UK). For detection of unlabeled primary antibodies, PE-conjugated rat anti-mouse IgG1, PE-conjugated goat anti-rat IgG (BD Pharmingen), or FITC-labeled mouse anti-rat IgG (Zymed; South San Francisco, CA), or biotinylated mouse monoclonal anti-rat
-chain (clone MRK-1; BD Pharmingen) antibodies were used, in conjunction with streptavidin-PE (BD Pharmingen) or extravidin-alkaline phosphatase (Sigma-Aldrich; Budapest, Hungary). The immunohistochemical (IHC) detection of fluorescein-labeled anti-B220 (BD Pharmingen) was performed using HRP-conjugated sheep anti-fluorescein IgG Fab (Roche; Mannheim, Germany).
Purification of T- and B-cells by MACS
Spleen cells (4 x 107) from (BALB/c x B6) F1 mice were divided into two aliquots and incubated with FITC-conjugated anti-B220 MAb or anti-MHC class II MAb on ice. After washing, the cells were incubated with anti-FITC beads (Miltenyi Biotec; Bergisch Gladbach, Germany), followed by separation using VarioMACS equipment. For B-cell enrichment, the B220-positive cells were collected using an LS column; for T-cell purification, the MHC class IIpositive cells were depleted with an LD column. After washing, 5 x 106 donor cells (B-, T-, and mixed) were injected IV into three different groups of SCID recipients.
Immunohistochemistry and Immunofluorescence
The single-label IHC staining of cryostat sections from various tissues with rat hybridoma supernatants containing IBL-10 and IBL-11 antibodies followed by biotin-amplified alkaline phosphatase detection was performed as described, using NBT/BCIP as chromogen substrate in the presence of 1 mg/ml levamisol (Balázs et al. 2001). For double, immunohistochemistry the visualization of stromal components by alkaline phosphatasebased labeling was followed by a short treatment of slides with dinitro-phenyl-hydrazine (1 mg/ml in PBS) to block endogeneous peroxidase activity. After rinsing, the slides were treated with normal rat IgG at 20 µg/ml to saturate the remaining binding sites of previously applied anti-rat Ig reagent, then incubated with FITC-conjugated anti-B220 MAb. The reaction was developed with HRP-conjugated sheep anti-FITC Fab fragments, using amino-ethyl carbasol as chromogen and 0.01% H2O2 as substrate in a 0.1 M Na-acetate buffer, pH 5.2.
For immunofluorescence, the sections were incubated with unlabeled primary antibodies followed by FITC-conjugated anti-rat IgG. For dual labeling, the sections were then blocked with 20% normal rat serum in PBS, followed by incubation with the second biotinylated MAb. After rinsing, the sections were incubated with streptavidinPE conjugate and viewed under an Olympus BX61 fluorescent microscope. The acquisition of digital pictures with a CCD camera and the morphometric image analysis were performed using the analySIS software.
Flow Cytometry
The reconstituted SCID mice were decapitated, and their blood was collected in tubes containing Na-heparin. Their spleens were removed and snap frozen for IHC evaluation. The undiluted blood was incubated with a cocktail of FITC-conjugated rat MAb against CD45, CD3, or anti-B220 antibodies and DaB1 MAb against H-2Kb on ice in the presence of 2.4G2 MAb (BD Pharmingen) to block FcRII-mediated binding of immunoglobulins. After incubation, the cells were washed with PBS containing 0.1% BSA and Na-azide. The DaB1 (mouse IgG1) alloantibody was revealed using PE-conjugated rat anti-mouse IgG1. After the lysis of erythrocytes, the cells were analyzed with a Becton-Dickinson FACSCalibur using the CellQuest software. The fluorescence distribution was determined from 10,000 electronic events gated on forward- and side-scatter parameters.
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Results |
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With the exception of intense reaction against the trabeculae by the IBL-10 MAb, there was no specific staining of red pulp reticular components (fibers or cells) by either the IBL-10 or the IBL-11 MAb. In the white pulp, the IBL-10 antibody labeled the adventitia of central arterioles and also some reticular cells in both the follicles and the PALS. The boundary toward the marginal zone was only faintly decorated, whereas in the border region between the T and B zones, a more-pronounced reaction could often be observed. Some more intensely labeled cells could also be detected in the deeper regions of follicles (Figure 1) . Induction of the germinal center by IP administration of SRBCs did not elicit the redistribution of IBL-10reactive cells in any region of the spleen (not shown).
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Unlike the generally even distribution of IBL-10positive reticular cells and fibers between follicles and PALS, IBL-11 MAb had a strong preference for T-cell zone reactivity (Figure 1). In addition, it also delineated an almost continuous rim within the follicles facing the white pulp proximal aspect of the marginal zone. The IBL-11positive cells did not expand beyond the white pulp distal layer of marginal sinus, as indicated by their position relative to the sinus lining cells expressing MADCAM-1 only (Figures 2C and 2D). Similarly to IBL-10, the IBL-11 MAb had no detectable reactivity against any lymphohemopoietic cell type isolated from spleen, lymph nodes, bone marrow, and thymus (not shown).
The IHC reactions indicated that whereas the follicular and marginal zone reactivities of IBL-10 and IBL-11 MAbs were clearly different, in the PALS they may be related. To confirm this interpretation, a dual immunofluorescent staining was performed. We found that the inner PALS that surrounds the central artery containing IBL-10positive adventitial cells appears to be dominated by IBL-11 single positive cells, while the outer PALS contains an IBL-10/11coexpressing compartment. In the follicles, the IBL-10positive cells seldom coexpress IBL-11, whose population is restricted to the cells adjacent to the marginal zone (Figures 2E and 2G).
Postnatal Development of the White Pulp Reticular Compartments Associated with Lymphocyte Colonization
Previous findings on the postnatal phase of spleen development have suggested the sequential reorganization of several stromal elements (including endothelial cells and FDC precursors) coupled with lymphocyte compartmentalization during the first 2 weeks after birth (Balázs et al. 2001; Balogh et al. 2001
). We tested whether the fibroblastic cells identifiable with IBL-10 and IBL-11 MAbs also change their tissue distribution during the establishment of adult-type architecture.
Immediately after birth (age <D1) there were only IBL-10positive reticular cells adjacent to the central artery, which at that stage was surrounded primarily by B-cells (identified as B220-positive lymphocytes). IBL-11 reactivity at this period could not be detected. A few days later (D35), a more-pronounced reactivity projecting toward the peripheral parts of the developing white pulp could be noted. By D7, a faint IBL-11 staining was observed, primarily located at the white pulp region distal to the central artery. At this stage, some focal accumulations of the B-cells could be noted, whereas the original ring-like cluster of B220-positive cells was dislodged from the immediate vicinity of the central artery by the gradual accumulation of T-cells. The border region between the T-cellrich area and these premature follicles was sometimes found to contain the IBL-10hi reticular cells mentioned previously. At D10, the IBL-11positive fibroblasts showed a tendency for follicular paucity in appearance, which was accompanied by the formation of a loose rim composed of IBL-11positive cells at the inner aspect of the MAdCAM-1positive marginal sinus, similar to the adult-type distribution (Figure 3) . The above sequence of events is summarized in Table 1.
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Next, we compared the ability of lymphocytes (either whole or purified T- and B-cells) to induce the rearrangement of fibroblast subsets reactive with IBL-10 and IBL-11 MAbs. The efficiency of cell transfer was evaluated by flow cytometric detection of donor H-2Kb alloantigen expression by the transplanted (B6 x BALB/c) F1 lymphocyte subsets among the total blood leukocytes. In contrast to the overwhelming majority of myeloid cells (granulocytes and monocytes), which appeared to be host derived, >90% of circulating lymphoid cells with lineage-restricted (B220 and CD3) differentiation markers expressed the donor-associated H-2Kb alloantigen in mice transplanted with pooled lymphocytes or with purified T- or B-cells, at a similar degree of donor cell load (Figure 5) . We found that 1 week after the cell transfer, the tissue distribution of IBL-10 and IBL-11positive fibroblast subsets was similar to that in normal mice, with some minor differences (Figure 6) . We could observe the appearance of IBL-10hi cells in the white pulp in all three groups of transplanted mice, and also the loosening of bundled IBL-11 cells at the peripheral parts of the white pulp, although the typical T-cell zone-associated reactivity of IBL-11positive fibroblasts could not be established. We also found that this partial remodeling of IBL-11positive reticular cells also occurred in recipients of F1 B-cells. The transfer of T-cells into SCID mice could also restore the presence of IBL-10hi cells, which are typically restricted to the follicular area in normal mice. In that respect, there was no difference between the abilities of mixed or purified T- or B-cells to induce formation of IBL-10hipositive fibroblasts and rearrangement of IBL-11. However, the zonal enrichment of the IBL-10hi cells at the periphery of follicles, as observed in normal mice, did not occur in T-celltransplanted SCID recipients because these cells were distributed evenly in the white pulp. Moreover, the tendency of IBL-11 cells to be arranged around the marginal zone aspect of follicles and within the PALS (thus leading to the paucity of these cells in the central part of the follicle) was also diminished, because these fibroblasts were distributed throughout the partially restored white pulp in these mice without any indication of gradient formation (Table 2). Permitting the tissue remodeling in these reconstituted SCID mice to continue for 2 more weeks could not correct this abnormal pattern of IBL-10hi and IBL-11 cell distribution (not shown).
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Discussion |
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With regard to the fibroblast components of the marginal zone area, it is interesting to note the close spatial relationship between the MAdCAM-1 reactivity of the sinus lining cells (Kraal et al. 1995) and the IBL-11 reactivity of adjacent fibroblasts. In humans, with no marginal sinus, the equivalent region, termed the perifollicular zone, contains MAdCAM-1positive fibroblasts, which exhibit a number of unusual features, including the expression of smooth muscle
-actin, smooth muscle myosin, cytokeratin 18, and VCAM-1 and Thy-1 antigens, and which are thought to be involved in regulating T-cell migratory events (Steiniger et al. 2001
). Although the MAdCAM-1 in unstimulated mice is primarily restricted to the sinus lining cells, its expression can also be observed at an extravascular location on FDCs in germinal centers, where the display of MAdCAM-1 antigen appears to delineate a continuous compartment originating from the marginal sinus (Balogh et al. 2002
). We found that this upregulation of MAdCAM-1 by germinal center stromal cells in SRBC-immunized mice is not accompanied by the appearance of IBL-10 or IBL-11 epitopes on the MAdCAM-1positive follicular cells in GCs, which suggests that the expression of these two markers is unrelated to the adaptation events of resting stromal elements to an altered lymphocytic microenvironment during GC formation.
The ability of transferred lymphoid cells to induce the appearance or redistribution of certain microenvironmental cells in immunodeficient SCID recipients has already been reported for FDCs and for reticular cells identifiable with WP-1 and RPSC-2 MAbs, respectively (Kapasi et al. 1993; Yoshida et al. 1993
). The preference for a certain lymphocyte subset presenting inducer molecules, such as LT
/ß2 or TNF
, appears to be lymphoid tissue specific because the lack of expression by B-cells has different degrees of inhibition of FDC differentiation in the spleen compared with that in lymph node or Peyer's patches (Tumanov et al. 2003
). In our studies on the spleen, the transfer of various lymphoid cells at the same number could effectively induce the rearrangement of IBL-11positive components, while it also initiated the appearance of IBL-10hi cells in the follicular region. Our data indicate that the IBL-11positive cells first appear at the peripheral part of the white pulp in both normal newborn and young adult SCID mice, but their subsequent rearrangement is probably related to the presence of mature lymphocytes and thus is impaired in SCID. It remains to be determined whether the remodeling of IBL-11positive cells from their original bundled distribution is due to their migration or simply to their separation caused by the lymphocyte colonization. It was interesting to note, however, that this remodeling also occurred in the B-cellreconstituted animals, despite the preferential T-zone expression of IBL-11positive reticular cells in normal mice. Similarly, the IBL-10hi cells in B-celltransplanted SCID mice also appeared at the same density as in mice reconstituted with T-cells, arguing against a special T- or B-cell requirement for the establishment of this subset, in contrast to the primarily B-celldependent differentiation of FDCs (Wang et al. 2001
). The failure to form an IBL-10hi fibroblast margin at the border between T- and B-cell zones in T-celltransplanted SCID recipients is probably related to the lack of B-cells. Therefore, we postulate that the B-cells (although effective inducers of their differentiation) probably restrict the infiltration of these cells into the deeper parts of the follicle, which was absent in these animals. Alternatively, the B-cells' role might be to direct the appearance of these (or similar cells) in the T-cell zone (Ngo et al. 2001
). It appears, therefore, that although the major events can overwhelmingly be corrected by the transfer of B-cells alone into young adult SCID mice, it is likely that the establishment of normal structure requires the availability of both subsets at an earlier period. To define precisely the entity of other stromal reticular cells influenced by lymphoid cells and their ontogenic relationship with each other, a systemic approach to delineate these various reticular cells with a battery of available monoclonal antibodies appears desirable.
In summary, our new monoclonal antibodies detect two reticular fibroblastassociated antigens whose tissue expression is restricted to two different compartments of splenic white pulp. Their appearances have different ontogenic features and requirements for antigen receptor bearing lymphocytes but they apparently display no particular lymphocyte preference for their induction and proper tissue distribution. Further analyses employing these new monoclonal antibodies in mutant mice with LT/TNF deficiencyrelated developmental abnormalities that affect the stromal architecture of peripheral lymphoid organs may further highlight the complex organization of the reticular fibroblast cells.
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Acknowledgments |
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We gratefully acknowledge the expert contribution of Ms Judit Melczer in maintaining the hybridoma cells, and Prof Béla Somogyi for access to the FACSCalibur flow cytometer at the Department of Biophysics. For rat MAbs IBL-10 and IBL-11, please contact PB.
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Footnotes |
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Literature Cited |
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