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The Journal of Immunology, 2008, 181, 1898 -1907
Copyright © 2008 by The American Association of Immunologists, Inc.
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Stromal Cells Confer Lymph Node-Specific Properties by Shaping a Unique Microenvironment Influencing Local Immune Responses1

Manuela Ahrendt*, Swantje Iris Hammerschmidt{dagger}, Oliver Pabst{dagger}, Reinhard Pabst* and Ulrike Bode2,*

* Functional and Applied Anatomy and {dagger} Institute of Immunology, Hannover Medical School, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103+ dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Lymph nodes (LN)3 are secondary lymphoid organs in which stimulation and activation of lymphocytes take place and effective immune responses are initiated. They are connected by the afferent lymphatics from the drained area. Therefore, the function of LN is to filter and monitor the lymph and obtain information about the infection state of the drained area (1, 2).

LN are highly compartmentalized and consist of a unique microarchitecture (2). However, the cellular composition of the B and T cells is dynamic due to the constant influx and efflux of these cells. Dendritic cells (DC) are loaded with Ag in the periphery and migrate via the afferent lymphatics into the draining LN. Naive T and B lymphocytes migrate into the LN preferentially via high endothelial venules (HEV) to screen for specific Ag presented by DC. Some stimulated T cells migrate into the B cell area to interact with Ag-specific B cells. Furthermore, activated T and B cells and their descendents leave the LN to spread throughout the body and re-enter the periphery where the specific Ag is present (1, 3).

In addition to these highly motile components, nonhematopoietic cells form a three-dimensional cellular network in the LN. These stromal cells mainly consist of fibroblastic reticular cells (FRC), which can be identified by the Ab ERTR-7 and are also positive for the glycoprotein podoplanin (gp38+). It is assumed that due to their interconnection, stromal cells form the skeletal structure of the LN, suggesting that these cells are not motile cells (4, 5, 6).

Both the immune cells and the stromal cells are able to produce soluble factors, e.g., cytokines and chemokines. For example, T lymphocytes, which are activated in response to the interaction with Ag-loaded DC, produce a set of cytokines including IL-2, IFN-{gamma}, and IL-4 (7). It was shown that stromal cells expressed CCL19/CCL21 within the T cell area and HEV of LN (6). Thus, CCR7-positive immune cells are able to migrate into the LN (8).

In addition, it was shown that within the LN immune cells interact with and migrate along the stroma to their compartments (9, 10). However, where and how the interaction takes place, is poorly understood.

The gut is continuously in contact with various numbers of different Ag, for example, food Ag, but also potentially pathogenic microorganisms, toxins, and other potentially harmful molecules. To protect the organism against these Ags, immune responses are initiated, resulting in tolerance or protective immune reactions. A characteristic feature of the gut immune system is a preferential response by producing IgA (11). Ag from the gut lumen is taken up by DC and transported into mesenteric lymph nodes (mLN), initiating an immune response in the gut (12, 13). This special location and function require an exclusive composition of cell subsets, cytokines, and chemokines, forming a unique microenvironment in mLN.

mLN-specific properties are as follows: MAdCAM-1 is expressed on HEV, but not on HEV of peripheral lymph nodes (pLN) (14, 15). Naive T cells, which enter the mLN via HEV, interact with DC arriving via the afferent lymphatics from the gut. These DC preferentially express the retinal dehydrogenase RALDH2 (16, 17). After activation, T cells and also B cells acquire a gut-homing phenotype, including high CCR9 and {alpha}4β7 integrin expression (17, 18, 19, 20). Furthermore, DC from the mLN differ from those in pLN in their ability to imprint T cells in the direction of Th2 (7, 21, 22, 23), e.g., IL-4 is found at a higher concentration in the mLN compared with the pLN (24). However, it is not known whether this unique microenvironment is due to the highly motile components of the mLN or to stromal cells.

Therefore, we established a model, in which a pLN regenerated in the draining area of the gut, which is ontogenetically occupied by the mLN. Under these in vivo conditions, we were able to study the influence and importance of the draining area and the stromal cells in the in vivo situation. For the first time, it has been studied whether LN-specific properties are influenced by the draining area or by stromal cells within the transplanted tissue.

To examine the functional relevance of the altered microenvironment in transplanted LN (LNtx), orally applied cholera toxin (CT) was used as an example of initiation of an immune response in the gut. CT is known as one of the most potent mucosal immunogens causing a strong intestinal IgA response after oral application (25, 26, 27, 28).

The data presented in this in vivo model document the active part played by stromal cells of LN in shaping a unique microenvironment, which is essential for local immune responses.


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

Rats from the standard inbred strains LEW/Ztm (RT.7a) and LEW.7B/Won (RT.7b) were bred and maintained at the central animal laboratory of the Hannover Medical School. The LEW.7B strain is identical to the congenic strain originally designated LEW.Ly1 (29). The RT system is a diallelic polymorphism of the CD45 molecular system. Male animals with a weight of 180–220 g at the beginning of the experiment were used for this study. Furthermore, female C57BL/6 and C57BL/6-Tg (ACTbEGFP) (designated here as EGFP mice) mice were bred at the central animal laboratory of the Hannover Medical School and were used at a weight of 18–25 g. All animal experiments were performed in accordance with the institutional guidelines and had been approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (no. 33-42502-05/960).

Intestinal surgery

The mLN and pLN were isolated from LEW.7B rats and EGFP mice and used as donors for LEW rats and C57BL/6 mice, respectively (Table I). Under the combined anesthesia with ketamine (Gräub) and Domitor (Pfizer), the mLN of the small and large intestine of the host were excised. The donor mLN or axillary and brachial LN were cut into small pieces (~10 mm3) and then transplanted into this region. These LN fragments were termed mLNtx and pLNtx, respectively. After 2, 4, 6, 8, 10, and 23 wk, 1 mg of BrdU/100 g of weight was given i.v. One hour later, the LNtx, mLNtx, or pLNtx were removed and analyzed (n = 4–5).


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  		Table I. Experimental design

 
Abs for flow cytometry

Cell suspensions from mLNtx and pLNtx were made and ~1 x 106 cells were incubated with biotinylated mAb W3/25 (CD4+ T cells) or OX8 (CD8+ T cells) and revealed by PerCP. B cells were characterized by OX12 and a PE-conjugated Ab was used as the secondary step. To differentiate between donor and host lymphocytes, mAbs His41 (FITC conjugated) was analyzed in the FACScan. DC and their subsets were classified as described previously (21). Isotype-matched mAb served as controls. All Abs were purchased from Serotec.

Quantification of mRNA expression among mLNtx and pLNtx

Total RNA of mLNtx, pLNtx, and control LN (n = 3) was isolated according to the manufacturer’s protocol (RNeasy Kit; Qiagen) and cDNA synthesis was performed with 50 mM oligonucleotide primer, 0.1 M DTT, 5x first strand buffer, 10 mM dNTP, 35 U/µl RNase inhibitor, and 200 U/µl Moloney murine leukemia virus reverse transcriptase (all obtained from Invitrogen) in a total volume of 20 µl at 37°C for 50 min. With this cDNA, quantitative real-time PCR was performed using the QuantiTect SYBR Green protocol from Qiagen. The primer sequences and amplicon sizes of IL-4 (5'-ATGTACCTCCGTGCTTGAAG-3' and 5'-TGAGCGTGGACTCATTCAC-3'; 117 bp), IL-2 (5'-CTGAAACTCCCCATGATGCT-3' and 3'-GAAATTTCCAGCGTCTTCCA-5'; 159 bp), IFN-{gamma} (5'-GCCCTCTCTGGCTGTTACTG-3' and 3'-CTGATGGCCTGGTTGTCTTT-5'; 221 bp), CCR9 (5'-GTGATTCCCCTGGCTCAGA-3' and 5'-CCCCACCAAAAGCTTAGTGA-3'; 200 bp), RALDH2 (5'-ACCTATCACCAGGCCTCCTT-3' and 5'-ACAAAATGGGGTTCATTGGA-3'; 174 bp), and GAPDH (5'-GATGACATCAAGAAGGTGGTGA-3' and 5'-ACCAGGAAATGAGCTTCACAAT-3'; 175 bp) for the housekeeping gene were used.

Immunohistochemistry

Cryostat sections of mLN control, mLNtx, as well as pLN and pLNtx (n = 4–5) were fixed in acetone:methanol solution (1:1, 10 min, –20°C). The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique was used to phenotype B lymphocytes (His14), donor lymphocytes (His41), and adhesion molecules such as HEV (His52, all obtained from Serotec) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1, Ost2; BD Biosciences) (30). After incubation with the primary Abs, slides were washed with TBST (0.05% Tween 20; Serva) and incubated with a bridging Ab (rabbit anti-rat; DakoCytomation) and then the APAAP complex (DakoCytomation) was applied. Fast Blue (Sigma-Aldrich) served as a substrate for alkaline phosphatase. Positive and negative controls produced the expected results. All sections were counterstained with hemalaun and mounted in glycergel (DakoCytomation). BrdU+ cells were determined also using the APAAP technique (24, 31).

Immunofluorescence histochemistry was performed according to standard protocols (32).

Briefly, sections were rehydrated in TBST (0.1 M Tris (pH 7.5), 0.15 M NaCl, and 0.1% Tween 20), preincubated with TBST containing 5% rat or mouse serum, and stained with fluorescent dye-coupled Abs (gp38-Cy5, ERTR-7-Cy3, meca79-Cy3, and LYVE-1-Cy3 (lymphatic vessel endothelial receptor 1)) in 2.5% serum/TBST. Nuclei were visualized by 4',6-diamidino-2-phenylindole staining (1 µg/ml 4',6-diamidino-2-phenylindole/TBST), and sections were mounted with Fluorescent Mounting Medium (DakoCytomation). Images were acquired using an Axiovert 200M microscope with Axiovision software (Zeiss).

Identification of T cells in the HEV of mLNtx and pLNtx

Cell suspensions were prepared from LEW.7B mLN. Then 50 x 106–100 x 106 cells were injected i.v. into LEW rats (n = 3) that had been transplanted 2 and 8 wk before (24). One hour later, the LNtx were removed and frozen in liquid nitrogen and stored at –80°C. Immunohistological staining of the HEV and the injected cells was conducted as described previously (24, 33).

Immunization

Purified CT (Sigma-Aldrich) was administered as described previously, with some modifications: Eight weeks after transplantation, LEW rats with mLNtx or pLNtx were immunized orally with 100 µg of CT (in 0.5 ml of 0.01 M PBS containing 0.2% gelatin) on days 0 and 14 (25). On day 19, the rats were exsanguinated, cell suspensions were made, and gut lavages were collected (n = 4–5). For that, the gut was cut into three equal pieces, each rinsed with intestinal lavage buffer (0.1 mg/ml trypsin inhibitor, 50 mM EDTA, and 0.1% BSA in PBS) and the supernatants were frozen immediately at –80°C. Analysis via flow cytometry and ELISA was performed as described below.

ELISA of gut lavage

The concentration of CT-specific IgA (CT-IgA) in the gut lavage of the transplanted rats after CT administration was analyzed in an ELISA (n = 5). The plates were coated with 0.1 µg/ml CT (Sigma-Aldrich) in PBS overnight at 4°C. After washing, the plates were blocked and samples were added undiluted or to a concentration of 1/512 and incubated for 90 min at 37°C. After washing, the detection Ab (biotinylated mouse-anti-IgA; BD Biosciences) was added and later detected with HRP (BD Biosciences), tetramethylbenzidine (BD Biosciences), and hydrogen peroxide (1:1) as the substrate. The reaction was stopped with 2 N H2SO4 (Merck). The OD was analyzed in an ELISA Reader (Bio-Tek Instruments).

Data analysis

Calculations, statistical analysis, and graphs were performed with the software GraphPad Prism 4.0. Statistical differences were calculated in the unpaired t test and are indicated by *, p < 0.05; **, p < 0.01; and ***, p < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
mLN or pLN fragments were transplanted into the mesentery after removing the mLN of LEW rats. The LN fragments were removed after different time points, an example of an early time point being 2 wk and of a late time point 8 wk after transplantation.

In LNtx fragments T and B cell areas were transiently disintegrated but regenerated within 8 wk

In LN of untreated animals (control LN), shown in Fig. 1A, the B cell areas were clearly identifiable and separated from T cell zones. Within the B cell areas, germinal centers were visualized by proliferation. Two weeks after transplantation, the T cell and B cell areas were destroyed and only small clusters of B and T cells were seen. Proliferation was distributed over the whole LN fragment and within the B cell clusters germinal centers were not identified (Fig. 1B). However, 8 wk after transplantation, B cells and T cells were found in typical compartments including germinal centers (Fig. 1C). A similar microanatomy was found 23 wk after transplantation, indicating that already after 8 wk the regeneration had been completed (Fig. 1D).


Figure 1
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  		FIGURE 1. The LN architecture is destroyed after transplantation, but regenerates within 8 wk. Cryosections of transplanted rat mLN and pLN were stained with mAbs against B cells (A–D, dark blue) and incorporated BrdU (A–C, red) (n = 4–5). The insets (right) show the area in the left box in a higher magnification. A, The mLN of an untreated animal shows a typical compartmental structure and germinal centers visualized by proliferation are present. B, Two weeks after transplantation, the architecture of the compartments is destroyed. Only small clusters of B and T cells are seen and germinal centers are absent. Eight weeks (C), but also 23 wk (D), after transplantation, large B and T cell areas are again found, comparable to those of the control mLN. In addition, germinal centers are present both in pLNtx and mLNtx.

 
Donor lymphoid cells emigrated from LNtx fragments

To characterize the lymphoid cell subsets in the LNtx fragments, cell suspensions were analyzed characterizing both the host and donor cells. At first, T and B cells within mLNtx and pLNtx were compared with a control mLN. Early after transplantation a significantly increased level of T cells was found which consisted of both CD4+ and CD8+ T cells (Fig. 2A), whereas B cells were decreased in mLNtx and pLNtx. However, 8 wk after transplantation, T as well as B cells showed no differences from the mLN control cell subset compositions any longer.


Figure 2
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  		FIGURE 2. Donor immune cells leave the LNtx fragments. A, mLNtx and pLNtx were analyzed for their cell populations by flow cytometry. After 2 wk, there was a relative increase of both CD4+ and CD8+ T cells compared with mLN of control animals. In contrast, there is a decrease in the B cells and DC in mLNtx as well as pLNtx compared with mLN control. Eight weeks after transplantation, the cell composition of both types of transplanted fragments is comparable to the mLN control. Means and SE are given (n = 3–5) and significant differences in the unpaired t test are indicated (**, p < 0.01 and ***, p < 0.001. B, After 2 wk, there was a significant decrease of these DC in mLNtx as well as pLNtx compared with mLN control. However, 8 wk after transplantation, both types of transplanted fragments no longer showed significant differences. Means and SE are given (n = 3–5) and significant differences in the unpaired t test are indicated (*, p < 0.05 and ***, p < 0.001. C, Donor lymphocytes of LEW.7B LNtx were detected within the LNtx over time by staining with the mAb His41 and analyzing via flow cytometry. mLNtx show significantly more donor cells than pLNtx 2 and 4 wk after transplantation. Means and SE are given from four independent experiments (significant differences in the unpaired t test are indicated (**, p < 0.01)).

 
In addition, MHCII+CD103+ cells were analyzed. This cell population, which was shown to be DC, demonstrated a highly decreased incidence within the transplanted fragments compared with DC in control mLN after 2 wk. However, in line with the T and B cells, the percentage of DC was comparable to those of the mLN control 8 wk after transplantation (Fig. 2B).

Finally, to observe how many donor cells remained in the LN fragments, the donor cells were quantified. Donor lymphoid cells within the mLNtx declined considerably at 2 and 8 wk after transplantation. Only 2.4% of mLNtx and 1.6% of pLNtx were identified as donor cells after 8 wk (Fig. 2C). However, a small population of migrated donor cells was found in secondary lymphoid organs, including the spleen of the recipients, at all time points (data not shown).

The subset composition of DC from pLNtx changed to that of mLN

DC from mLN differed from those of pLN based on the expression levels of MHC class II (MHCII) and CD103 (Fig. 3). In the pLN MHCIIhighCD103– DC were found, whereas in the mLN MHCIIhighCD103– cells were completely absent. In contrast, MHCII+CD103+ cells were preferentially seen in the mLN (Fig. 3). Only mLN-specific DC subsets (MHCII+CD103+) but not MHCIIhighCD103– DC were found in mLNtx and also in pLNtx (Fig. 3), demonstrating that the subset composition of pLN DC changed to that of mLNtx.


Figure 3
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  		FIGURE 3. Eight weeks after transplantation, DC of pLNtx exhibited the phenotype of mLN DC. mLNtx and pLNtx were analyzed by gating on the T and B cell-negative population for their MHCII+CD103+ DC population by flow cytometry. Dot plots from DC of the mLN and pLN control as well as mLNtx and pLNtx are shown. In the control mLN and also in mLNtx and pLNtx, MHCII+CD103+ DC were found (region II), whereas MHCII highCD103– cells (region I) were only detected among the population of control pLN DC. The dot plots represent three independent experiments.

 
In addition, in control animals, CD11c expression was different among pLN MHCIIhighCD103– and mLN MHCII+CD103+ cells. Similar to the pattern of the DC subset composition in pLNtx, the CD11c expression was comparable to that of the mLN control (mLN control, 65%; pLNtx, 64 ± 4.4%, n = 3).

Stromal cells survived after transplantation

To analyze whether stromal cells also disappeared from the transplanted fragments, mLNtx and pLNtx of EGFP+ mice were transplanted into C57BL/6 mice and after 8 wk the LN were removed. GFP+ cells were identified in the B cell and T cell areas (Fig. 4, A and B). As was expected from previous results in the rat model, most of the T and B cells were GFP negative, showing that most lymphocytes originated from the host.


Figure 4
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  		FIGURE 4. FRC survive after transplantation. A–H, LN of EGFP+ mice (green) were transplanted into C57BL/6 mice and removed 8 wk later (n = 3). A and B, B and T cell subsets of the LNtx fragments were analyzed by fluorescent staining. GFP+ cells are seen in both the B cell area (A, red) and in the T cell area (B, red). The inset shows a typical B cell follicle (red) in the B cell area. Although GFP+ immune cells disappeared from the regenerated LN fragment, stromal cells survived and are seen as remaining GFP+ cells. C–H, Stromal cells of the transplanted fragments were analyzed by immunofluorescent staining with gp38 and ERTR-7. Donor cells (green) were found within mLNtx as well as pLNtx (C and D). E, Staining with gp38 (blue) and F, staining with ERTR-7 (red). The merging of C and E and also of D and F shows that most FRC are GFP+ (G and H.). Nearly all gp38 and ERTR-7+ cells are GFP+.

 
Furthermore, the transplanted GFP+ LN fragments were stained with the FRC marker gp38 (Fig. 4E) and with the fibroblastic marker ERTR-7 (Fig. 4F). In contrast to the lymphoid cells, most gp38+ and ERTR-7+ cells were also GFP+ (Fig. 4, C–H), demonstrating that most of these stromal cells survived after transplantation. This was seen in mLNtx as well as in pLNtx. Interestingly, most of the follicular DC were also GFP+ (data not shown), indicating that these cells survived too.

Taken together, complete removal of the mLN and insertion of donor mLN/pLN resulted in destruction of the LN architecture, which was regenerated at 8 wk after transplantation. Therefore, the 8-wk time point was chosen to analyze the microenvironment of regenerated LN fragments.

Reconnection of the LNtx fragments to the blood stream

To analyze the presence of HEV, the LNtx fragments (mLNtx and pLNtx) were stained with a marker for HEV immunohistologically. Within mLNtx as well as pLNtx, HEV were identified (Fig. 5A).


Figure 5
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  		FIGURE 5. The LNtx fragments are connected to the blood vessels and lymphatics. Eight weeks after transplantation, mLNtx and pLNtx were excised and the presence of HEV (A–D) and lymphatics (E–H) was analyzed. A, Lymphocytes of the congenic LEW.7B strain were injected into transplanted LEW rats, which were exsanguinated 1 h after injection. Cryosections of the transplanted mLN and pLN fragments (LNtx) were stained for HEV (light blue) and donor cells (dark blue). The inset shows a higher magnification. Injected cells are found in the LNtx and in the wall of the HEV (n = 3) B, LN fragments of EGFP+ mice were transplanted into C57BL/6 mice and 8 wk after transplantation mLNtx and pLNtx were removed and the HEV were identified immunohistologically. In both LNtx GFP+HEV+ cells were found (B–D). E, Eight weeks after transplantation, the dye Berlin blue was injected into the subserosa of the gut. Berlin blue can only transported via the lymph. The dye is found in the lymph vessels and also in the sinus of the LNtx. The inset shows a higher magnification. F–H, LN fragments of EGFP+ mice were transplanted into C57BL/6 mice and 8 wk after transplantation mLNtx and pLNtx were removed and stained for lymphatic endothelial cells (LYVE-1; n = 2). LYVE-1 was present in the marginal lymphatic sinus in pLNtx and also in mLNtx. Most of these cells are GFP+ (E–H).

 
To investigate whether these HEV were reconnected to the blood stream and fulfilled their physiological function, naive mLN lymphocytes from the congenic rat strain (LEW.7B) were isolated and injected i.v. One hour later, the donor cells were found in the wall of the HEV. Then, EGFP+ mice were transplanted into C57BL/6 mice and after 8 wk the transplants were removed and the HEV were stained (Fig. 5, B–D). In these LNtx fragments, GFP+ HEV were found, documenting the survival of the donor HEV (Fig. 5, B–D).

Reconnection of the afferent lymphatics

To ensure that not only the blood vessels were connected to the LNtx fragments, the presence of lymphatics was also checked 8 wk after transplantation. Berlin blue was injected into the subserosa of the gut and it was found in the LN fragments as transported via afferent lymphatics to lymph sinuses of mLNtx as well as pLNtx (Fig. 5E).

Lymphatic endothelial cells visualized by a staining against the lymphatic endothelial hyaluronan receptor (LYVE-1) within the LNtx fragments were seen (Fig. 5, F–H). LYVE-1+ cells of the sinus are mostly GFP+, indicating that these cells also survived during regeneration (Fig. 5H).

MAdCAM-1 was not expressed in pLNtx

To identify MAdCAM-1 expression, mLNtx and pLNtx were stained immunohistologically and compared with a control mLN and control pLN. The adhesion molecule MAdCAM-1 was expressed on HEV within the mLN, but not on HEV in pLN (Fig. 6, A and B). Similarly, MAdCAM-1 expression was found in mLNtx (Fig. 6C) but was absent in pLNtx (Fig. 6D).


Figure 6
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  		FIGURE 6. MAdCAM-1 expression is not influenced by the drained area. MLN (A) and pLN (B) of control animals as well as mLNtx and pLNtx were analyzed by immunohistological staining against MAdCAM-1 8 (C and D) and 23 wk (E and F) after transplantation. MAdCAM-1 was detected in the control mLN as well as the mLNtx but not in the pLN control. Over a period of 23 wk, MAdCAM-1 expression was not detected in pLNtx (F) (n = 4–5). The insets show a higher magnification.

 
This expression pattern was seen at all time points measured (Fig. 6, E and F).

The drained area influenced IL-4 expression, whereas the expression pattern of CCR9 and RALDH2 was LN specific

To analyze whether stromal cells or the drained area influenced the cytokine pattern within the LN, various cytokines were quantified via real-time PCR. The expression of IL-4, IL-2, and IFN-{gamma} mRNA differed among mLN and pLN of control animals (34). In detail, IL-4 mRNA was found more in mLN than in pLN. In contrast, IL-2 and IFN-{gamma} mRNA were preferentially detected in pLN.

The IL-4, IL-2, and IFN-{gamma} mRNA in the LN fragments of untreated (under steady-state conditions) and CT-treated animals (immune response) were studied. The different expression of IL-4 in untreated animals was no longer detectable between mLNtx and pLNtx, indicating that IL-4 expression was influenced by the drained area (Fig. 7A). In contrast, IFN-{gamma} and IL-2 showed more expression in the pLNtx under steady-state conditions, demonstrating that the expressions of these cytokines were not controlled by the drained area. The differences were increased when CT was given (Fig. 7A).


Figure 7
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  		FIGURE 7. The expression pattern of Th1 cytokines, CCR9, and RALDH2 is LN specific. Eight weeks after transplantation, CT was given orally. The mLNtx and pLNtx were excised, mRNA was isolated, and real-time PCR in triplets was performed. Untreated mLNtx and pLNtx were also analyzed. The data are normalized to the housekeeping gene GAPDH for rats and are given from three animals. Significant differences in the unpaired t test are indicated (*, p < 0.05 and **, p < 0.01). A, Eight weeks after transplantation, IL-4 was detected in both types of transplanted fragments on similar expression levels and at a higher amount after CT treatment. In contrast, the expression of IFN-{gamma} and IL-2 was significantly increased in pLNtx compared with mLNtx in untreated animals. After CT treatment, both cytokines were strongly up-regulated in mLNtx and to a greater extent in pLNtx. B, CCR9 expression in mLNtx and pLNtx was expressed similarly compared with the mLN and pLN control, respectively. The same is valid for RALDH2 expression.

 
It has been shown that retinal dehydrogenases RALDH1–3 are important for the induction of gut homing receptors. RALDH2 is preferentially expressed in the mLN (16). To test whether this enzyme was dependent on the drained area, RALDH2 expression was quantified via real-time PCR in the LN fragments. The data showed that RALDH2 in mLNtx was expressed similarly compared with the mLN control. In contrast, the RALDH2 expression in pLNtx was at a low level similar to level of the pLN control (Fig. 7B).

Gut tropic T and B cells are positive for the chemokine receptor CCR9 which is influenced by retinoic acid, the product of RALDH2 (35, 36). Therefore, the CCR9 expression of LN transplants was analyzed. The same pattern was seen for CCR9: The expression in mLNtx was comparable to the mLN control, whereas pLNtx were as low as the pLN control (Fig. 7B). The data document that CCR9 and RALDH2 expression were not influenced by the drained area.

The IgA response in pLNtx was lower than in mLNtx-transplanted rats

Finally, it was analyzed whether these differences in pLNtx influenced a typical gut immune response induced in the draining area of the LNtx fragments. Therefore, the B cell phenotype and response including CT-specific IgA Abs, were investigated after an oral dose of CT.

The percentage of surface IgA+ B cells was much lower in pLNtx compared with mLNtx (Fig. 8A). CCR9 expression was decreased on the pLNtx B cells compared with those of mLNtx (Fig. 8B). This indicates that gut-specific B cells were reduced in pLNtx after CT treatment.


Figure 8
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  		FIGURE 8. CCR9+ B cells were less increased in pLNtx after CT administration. Eight weeks after transplantation, CT was administered and mLNtx and pLNtx were excised. A, Cell suspensions were made and IgA+ B cells were analyzed by flow cytometry. The percentage of surface IgA+ B cells was much lower in pLNtx compared with mLNtx. B, B cells were separated via positive selection using the MACS technique, mRNA was isolated, and real-time PCR in triplets for CCR9 was performed. The data are normalized to the housekeeping gene GAPDH for rats and are given from two animals. Significant differences in the unpaired t test are indicated (*, p < 0.05). CCR9 expression was lower among the pLNtx B cells compared with those of mLNtx C, The gut was lavaged to perform a CT-specific IgA ELISA. The data show significantly higher CT-specific IgA levels in mLNtx than in pLNtx. Means and SE are given from five to eight independent experiments (significant differences in the unpaired t test are indicated (**, p < 0.05)).

 
As a result of this reduction, CT-specific IgA Abs were also reduced in pLNtx animals (Fig. 8C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been shown that LN transplanted into the s.c. layer of the skin were reconnected to the afferent lymphatics (37, 38, 39). However, when a LN was transplanted into the omentum of pigs the LN disappeared and did not regenerate (38). In the present study, it was documented for the first time that LN fragments implanted into the mesentery of mice and rats were regenerated and reconnected to the lymphatic system independent of their origin. Previously, another group suggested that the blood supply of the LNtx alone is not sufficient to establish the LN in this area (38). In our in vivo model, both the afferent lymphatics and the blood supply were also regenerated, documented by functional tests in pLNtx and mLNtx. Since these two routes are entry sites of immune cells and soluble molecules, reconnection to the draining area and the blood is a prerequisite of a complete functional regeneration. The present study shows that LNtx fragments were initially destroyed but regeneration was completed 8 wk after transplantation. This period of regeneration was confirmed in recently published data (40).

The donor immune cells disappeared during the first 2 wk. The host immune cells were able to enter the LN fragments, probably due to the regenerated blood and lymph supply, resulting in a resettlement of the compartments. One type of these highly motile cells are the DC, which migrate from the draining area into the LN. DC from mLN can be distinguished from those from pLN by their different subset composition and expression pattern of surface molecules (e.g., CD103) (21, 22). The results showed that DC from pLNtx had been replaced by DC with a subset composition and phenotype typical for mLN, underlining the generally accepted observation that the DC came from the drained area.

In contrast to the highly motile immune cells, nonhematopoietic cells survived and were not replaced during regeneration of the LNtx. This aspect has not been described before. Nonhematopoietic cells form a three-dimensional network building the backbone of the LN. One type of these resident cells forms HEV, which play a critical role in recruiting lymphocytes into LN by expressing homing molecule receptors, e.g., MAdCAM-1 and pLN addressins, and presenting chemokines from the draining area (15, 41). The current study illustrates that HEV not only survived during regeneration but also retain their site-specific expression pattern (such as the absence of MAdCAM-1 expression in pLNtx). This was independent of the reconnected lymph supply which transports Ags and low-weight molecules, e.g., chemokines, from the gut via the conduits to the lumen of HEV.

Another cell population of these nonhematopoietic cells are fibroblastic reticular cells (FRC) identified here by gp38 and ERTR-7 (5, 6). FRC are able to regulate the entry of CCR7+ immune cells via CCL19 and CCL21 expression (5, 42), the entry and location of naive T and B cells within the paracortex of the LN (9), and to produce survival factors which influence naive T homeostasis (6). Finally, it is known that FRC form a cellular sleeve around the conduits by anchoring themselves to the basement membrane and are therefore important structural elements of the LN architecture (43, 44). However, little is known whether FRC as a stromal cell population influence immune responses within the LN.

Our investigations show that stromal cells are involved in the expression pattern of cytokines. During an immune response, cytokines are relevant molecules to drive T cell polarization into a distinct direction. For example, IL-4 is a Th2 cytokine, which is preferentially found in the mLN (34). Our data show that the expression level of IL-4 was influenced by the drained area in steady-state conditions, but also during an immune response. In contrast, IL-2 and IFN-{gamma} were found to be expressed preferentially in the pLN and their drained area (34). The pattern of IL-2 and IFN-{gamma} expression in the pLNtx is unaffected. However, whether the stromal cells themselves express, for example, IL-2, or whether they indirectly influence the motile components to up-regulate these cytokines is not known.

Another important aspect of the present study is that RALDH2 expression in pLNtx was only marginally similar to the situation in control pLN. Retinal dehydrogenases RALDH1–3 are important for the induction of gut-homing receptors. RALDH2 is preferentially expressed on DC in the mLN and is not detectable in the pLN (16, 45). Thus, since DC in the pLNtx are host derived, only the stromal cells of the transplanted pLNtx are able to influence indirectly the expression of RALDH2 on these immigrated DC or directly fail to express RALDH2. Preliminary data indicate that FRC within the mLN itself express RALDH2, whereas FRC within the pLN fail to express RALDH1–3 (data not shown).

In response to retinoic acid, T and B cells within the mLN are found to express CCR9 and {alpha}4β7 integrin. Thus, activated T and B cells are imprinted with the gut-homing phenotype to re-enter the gut via their specific expression pattern (19, 46, 47).

Our study shows that pLNtx exhibits an increased number of B cells, expressing minor levels of CCR9. It strongly suggests that the failed RALDH2 expression of pLNtx leads to a decreased CCR9 expression on B cells after initiating an immune response. The functional consequence of this different expression pattern of pLNtx is a lower IgA titer against orally applied CT as seen in mLNtx. Thus, the function of the pLNtx is disturbed, indicated by an impaired immune reaction against Ags coming from the gut.

Taken together, LN consist of both highly motile immune cells and resident nonhematopoietic stromal cells forming the backbone of the LN. When a peripheral LN was implanted into the mesentery, the highly motile cells disappeared from the LNtx, but the skeletal backbone survived after transplantation. These stromal cells seem to be able to reprogram the highly motile cells in educating them to the organotypical expression pattern. This leads to the situation that a regenerated pLN despite being perfused by the gut lymph still has the LN-specific properties of a pLN. The function of this LN is disturbed, indicated by decreased IgA cells and specific Abs against orally administered Ag, e.g., CT. Thus, stromal cells, as an organotypical skeleton of lymphoid organs, seem to be important for an efficient immune response.


   Acknowledgments
 
The comments of Jutta Schade have been of great help. We also thank Nadja Thiessen and Anika Hahn for help with the operations, Frauke Weidner for excellent technical assistance, and Sheila Fryk for correction of the English.


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


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

1 This work was supported by the German Research Foundation (SFB621/A10). Back

2 Address correspondence and reprint requests to Dr. Ulrike Bode, Anatomie II, OE 4120, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail address: Bode.Ulrike{at}MH-Hannover.de Back

3 Abbreviations used in this paper: LN, lymph node; CT, cholera toxin; FRC, fibroblastic reticular cell; MAdCAM-1, mucosal addressin cell adhesion molecule 1; mLN, mesenteric LN; mLNtx, transplanted mLN; pLN, peripheral LN; pLNtx, transplanted pLN; PP, Peyer’s patch; RALDH, retinal dehydrogenase; MHCII, MHC class II; APAAP, alkaline phosphatase anti-alkaline phosphatase; LYVE-1, lymphatic vessel endothelial receptor 1. Back

Received for publication January 10, 2008. Accepted for publication May 23, 2008.


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 Introduction
 Materials and Methods
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 Disclosures
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S. I. Hammerschmidt, M. Ahrendt, U. Bode, B. Wahl, E. Kremmer, R. Forster, and O. Pabst
Stromal mesenteric lymph node cells are essential for the generation of gut-homing T cells in vivo
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