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Originally published online as doi:10.1189/jlb.0407213 on June 28, 2007

Published online before print June 28, 2007
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(Journal of Leukocyte Biology. 2007;82:869-876.)
© 2007 by Society for Leukocyte Biology

Low-density cells isolated from the rat thymus resemble branched cortical macrophages and have a reduced capability of rescuing double-positive thymocytes from apoptosis in the BB-DP rat

Vinod Sommandas*,1, Elizabeth A. Rutledge{dagger}, Brian Van Yserloo{dagger}, Jessica Fuller{dagger}, Åke Lernmark{dagger} and Hemmo A. Drexhage*,{dagger}

* Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; and
{dagger} Department of Medicine, R.H. Williams Laboratory, University of Washington, Seattle, Washington, USA

1 Correspondence: Dept. of Immunology, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: v.sommandas{at}erasmusmc.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biobreeding-diabetes prone (BB-DP) rats spontaneously develop organ-specific autoimmunity and are severely lymphopenic and particularly deficient in ART2+ regulatory T cells. A special breed, the so-called BB-diabetic-resistant (DR) rats, are not lymphopenic and do not develop organ-specific autoimmunity. The genetic difference between both strains is the lymphopenia (lyp) gene. Intrathymic tolerance mechanisms are important to prevent autoimmunity, and next to thymus epithelial cells, thymus APC play a prominent part in this tolerance. We here embarked on a study to detect defects in thymus APC of the BB-DP rat and isolated thymus APC using a protocol based on the low-density and nonadherent character of the cells. We used BB-DP, BB-DR, wild-type F344, and F344 rats congenic for the lyp gene-containing region. The isolated thymus, nonadherent, low-density cells appeared to be predominantly ED2+ branched cortical macrophages and not OX62+ thymus medullary and cortico-medullary dendritic cells. Functionally, these ED2+ macrophages were excellent stimulators of T cell proliferation, but it is more important that they rescued double-positive thymocytes from apoptosis. The isolated thymus ED2+ macrophages of the BB-DP and the F344.lyp/lyp rat exhibited a reduced T cell stimulatory capacity as compared with such cells of nonlymphopenic rats. They had a strongly diminished capability of rescuing thymocytes from apoptosis (also of ART2+ T cells) and showed a reduced Ian5 expression (as lyp/lyp thymocytes do). Our experiments strongly suggest that branched cortical macrophages play a role in positive selection of T cells in the thymus and point to defects in these cells in BB-DP rats.

Key Words: type I diabetes • autoimmunity • positive selection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biobreeding-diabetes prone (BB-DP) rats spontaneously develop autoimmune insulitis, autoimmune thyroiditis, and other organ-specific autoimmunity. The advantage of studying the BB rat model of organ-specific autoimmunity is the involvement of a well-defined, thymus-derived regulatory T (Treg) cell population. These Treg cells express the molecule ART2 [1 ]. BB-DP rats are severely lymphopenic and particularly deficient in ART2+ Treg cells [2 3 4 5 6 ]. A special breed of BB rats, the so-called BB-diabetic-resistant (DR) rats, are not lymphopenic and do not develop organ-specific autoimmunity, because of the presence of ART2+ Treg cells. Depletion of these cells in the BB-DR rat induces organ-specific autoimmunity [7 ], and transfer of these cells obtained from BB-DR rats into BB-DP rats prevents organ-specific autoimmunity [8 ]. Recently, this ART2+ Treg cell population was investigated in more detail, and it was shown that one subset was CD4+CD25–Foxp3–PD-1+ART2+ and this population was as capable of preventing the development of diabetes in the BB-DP rat as the presently well-established CD4+CD25+Foxp3+PD-1+ART2+ Treg subset [9 ].

The genetic difference between the BB-DP rat and the BB-DR rat is the lymphopenia (lyp; iddm2) gene, which is a major rat diabetes susceptibility gene located on chromosome 4. Homozygosity for the defective lyp gene, as is the case in the BB-DP rat, leads to the severe T lymphocytopenia and the lack of ART2+ Treg cells [10 , 11 ]. Recently, the lyp gene (also called Ian5, Gimap5, Ian4L) was found to carry a frame-shift mutation and a severe truncation of the protein [11 , 12 ]. Ian5 is thought to be involved in apoptosis, being associated with the antiapoptotic proteins Bcl-2 and Bcl-xL [12 13 14 ].

Intrathymic tolerance mechanisms are important mechanisms to prevent organ-specific autoimmunity. Next to thymus epithelial cells, thymus APC, i.e., dendritic cells (DC) and macrophages, play a prominent role in these central tolerance mechanisms. It is interesting that thymus transplantation experiments by Georgiou et al. [15 16 17 ] showed a pivotal role of defects in antigen presentation by bone marrow (BM)-derived cells in the thymus of BB-DP rats. These defects in antigen presentation were crucial for the T cell lymphopenia of the rat and the ultimate development of organ-specific autoimmunity.

A direct, in vitro approach has not been used to study the defects of thymus BM-derived APC in the BB-DP rat, and we embarked on such a study. We isolated APC from the rat thymus using a protocol based on the low-density and nonadherent character of the cells, as this protocol is known to result in a population of APC, previously identified as thymus DC on the basis of their dendritic morphology, expression of MHC Class II molecules in the absence of T and B cell markers, and their excellent T cell stimulatory capacity [18 , 19 ]. The low-density cell (LDC) isolation is a well-accepted method to isolate spleen DC and is in use since the early days of DC studies [20 ]. In our experiments, BB-DP and BB-DR rats of the Seattle subline and wild-type F344 rats and F344 rats congenic for the region of the BB-DP rat chromosome 4, on which the lyp gene is located, were used. BB-DP/Seattle rats are lymphopenic, and autoimmune diabetes occurs in 100% of the Seattle BB-DP rats. Congenic F344 rats homozygous for the lyp region of the BB-DP rat chromosome 4 (designated "F344.lyp/lyp rats") have a similar extent of T cell lymphopenia and other cellular abnormalities as the BB-DP rat, including nearly absent ART2+ T cells [21 , 22 ]. The rats do, however, not develop organ-specific autoimmunity, as they lack other important iddm genes for such development on other chromosomes.

We here describe that thymus LDC are phenotypically rather reminiscent of branched cortical macrophages than of thymus medullary and cortico-medullary DC. The population of thymus LDC of the BB-DP and the F344.lyp/lyp rat had a strongly diminished capability of rescuing (double-positive) thymocytes from apoptosis and a reduced Ian5 expression as compared with such LDC of nonlymphopenic rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male and female BB-DP, BB-DR, F344.+/+, and F344.lyp/lyp rats were from the Seattle colony. Wistar rats were purchased from Harlan (Zeist, The Netherlands). All rats were kept under controlled light conditions (12/12 h light/dark cycle) throughout this study. A standard pelleted diet (AM-11, Hope Farms BV, Woerden, The Netherlands) and tap water were provided ad libitum. One hundred percent of the BB-DP rats became diabetic. BB-DP rats were tested daily for glucosuria (Gluketur test sticks, Boehringer Mannheim BV, Almere, The Netherlands). For all experiments, age-matched rats were used.

Cell preparations
Thymus LDC, spleen LDC, spleen macrophages, and BM-derived DC
Thymus LDC and spleen LDC were enriched according to the method of Knight et al. [20 ] and Delemarre et al. [23 ] with slight modifications. Briefly, thymi and spleens from BB-DP/BB-DR and F344 rats were minced and digested for 1 h at 37°C in RPMI-1640 medium (Life Technologies, Breda, The Netherlands) with 25 mM glutamax-1 and 25 mM HEPES (hereafter referred to as RPMI+) containing 125 U/ml collagenase (type III, Worthington Biochemical, Freehold, NJ, USA) and 0.1 mg/ml DNase (Boehringer Mannheim BV).

The remaining tissue was teased through a 105-µm filter, and the erythrocytes were removed by lysis. Finally, the separated cells were washed and cultured in RPMl+ supplemented with 10% inactivated FCS (FCSi), penicillin (100 U/ml, Seromed, Biochrom, Berlin, Germany), and streptomycin (0.1 mg/ml, Seromed). After an overnight culture period in culture flasks (Costar Europe, Badhoevedorp, The Netherlands; 37°C, 5% CO2 incubator), the nonadherent cells and (in the case of the spleens) the adherent cells were harvested. The spleen adherent cells were for over 95% macrophages. LDC were isolated from the nonadherent cells by using a 14.5% (w/v) Nycodenz (Nycomed Pharma As, Oslo, Norway) density gradient (800 g for 20 min). LDC were collected from the interphase and washed.

BM precursor-derived DC were obtained as described in detail previously [24 ].

Antibodies
In flow cytometric analysis, we used anti-MHC class II conjugated to PE (1:400, MRC OX6, Serotec, UK), anti-B7-1 (undiluted, CD80, BD PharMingen, Flanders, NJ, USA), anti-B7-2 (undiluted, CD86, BD PharMingen), anti-CD40 (1:10, BD PharMingen), anti-rat DC-FITC (undiluted, MRC OX62, Serotec), ED1 (1:10, DC, monocytes, macrophages, Serotec), ED2/anti-CD163 (1:100, macrophages, Serotec), and ED3/anti-CD169 (1:1000, Serotec).

In immunohistochemistry, we used anti-MHC class II (1:5, Serotec), anti-rat DC/OX62 (1:10, Serotec), and ED2/anti-CD163 (1:20, Serotec). All were conjugated to biotin.

Flow cytometric analysis
Flow cytometric analysis was performed via standard procedures and as described previously [24 ].

Immunocytology
Cytocentrifuge preparations were made of the isolated thymus LDC fractions and fixed in acetone (Fluka, Buchs, Germany) for 10 min (endogenous peroxidase was blocked by adding 0.05% H2O2) and thereafter, incubated with normal rabbit serum (Dako, Glostrup, Denmark), 10% in PBS for 10 min before incubating for 1 h with the biotinylated anti-MHC class II antibody. After washing three times with PBS, the slides were incubated with streptavidin-biotin complex conjugated with alkaline phosphatase (Dako). The slides were rinsed three times in PBS and stained with Fast Blue BB base (Sigma, Zwijndrecht, The Netherlands). Counterstaining was not performed. Finally, the slides were rinsed in water and mounted in Kaiser's gelatine (Merck, Rahway, NJ, USA).

For double-staining, fixation and subsequent incubation with the first mAb, followed by a rabbit anti-mouse antiserum conjugated with HRP, were performed. After blocking with normal mouse serum (10%), the slides were incubated with a directly biotinylated second mAb. Slides were rinsed three times in PBS and incubated with streptavidin-biotin complex conjugated with alkaline phosphatase (Dako). Slides were stained sequentially with Fast Blue BB base (Sigma) and 3-amino-9-ethylcarbazole (AEC; Sigma). Finally, the slides were rinsed in water and mounted in Kaiser's gelatine (Merck).

Immunohistology
mAb against ED2 and OX62 were used to stain histological sections of the thymus. As a control, we used nonspecific murine IgG1. Histological preparations were fixed in acetone (Fluka) for 10 min (endogenous peroxidase was blocked by adding 0.05% H2O2) and thereafter, incubated with normal rabbit serum (Dako), 10% in PBS for 10 min before incubating for 1 h with the appropriate mAb. Subsequently, the slides were incubated with rabbit anti-mouse antiserum conjugated with HRP for AEC staining. Finally, the slides were rinsed in water and mounted in Kaiser's gelatine (Merck).

Methods for quantitative PCR (qPCR) for Ian5 expression
qPCR for Ian5 expression was performed as described in detail previously [24 ].

MLR
T cells from Wistar rats were enriched using a nylon wool column as described in detail previously [24 ]. In short, spleens were minced and teased through a 105-µm filter, and the erythrocytes were removed by lysis. Cells were washed and loaded onto a nylon wool column (3 g, Polyscience, Eppelheim, Germany). After 1 h incubation, T cells (80–90% CD5+ cells) were harvested by collecting the effluent. For the MLR, APC from the different rats were added at various ratios to T cells (fixed number of 150,000 T cells/well) in flat-bottom, 96-wells plates (Nunc, Roskilde, Denmark). Subsequently, these cells were cultured for 3 days in RPMI+ medium including FCSi and antibiotics. In the MLR, T cell proliferation was measured via tritiated thymidine ([3H]TdR) incorporation. Radioactivity was counted in a liquid scintillation analyzer (LKB Betaplate, Wallac, Turun, Finland).

Thymocyte apoptosis assay
Thymocytes were isolated from the high-density fraction after overnight culture (macrophages and residual epithelial cells attach to the bottom of the flasks). These thymocytes were thereafter cocultured with thymus LDC in flat-bottom, 96-wells plates (Nunc) at a ratio of 2:1. After harvesting the cells, the capacity of thymus LDC to rescue double-positive thymocytes from apoptosis was assessed by 7-amino-actinomycin D (7-AAD) staining using a flow cytometer (FACSCalibur, Becton Dickinson, Sunnyvale, CA, USA) gating on the CD4+CD8+ cells. For determination of background staining, cells were incubated with labeled, irrelevant antibodies or with secondary antibodies.

Statistical analyses
Statistical analyses were performed using the nonparametric Mann-Whitney test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The phenotype of thymus LDC strongly suggests that the cells represent the thymus branched cortical macrophages
Analyzing the thymus LDC population with DC and macrophage-specific markers, we found that the thymus LDC were, in particular, positive for the macrophage marker ED2/CD163: 83% (median, range 76–90%, n=3) of the cells were positive in flow cytometric analysis in a dim-to-intermediate-staining manner (Fig. 1A and 1F ), and in immunocytology, approximately 80% of the LDC were clearly positive (Fig. 1C and 1D) . ED2/CD163 (a member of the scavenger receptor cysteine-rich group B family, functioning as a scavenger receptor for hemoglobin-haptoglobin complexes) identifies most subpopulations of mature tissue macrophages, including spleen red pulp macrophage, thymus cortical macrophages, Kupffer cells in the liver, resident BM macrophages, and CNS perivascular and meningeal macrophages, but is absent on monocytes [25 ].


Figure 1
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  		Figure 1. (A) Dot plots of thymus LDC with ED2 expression on the x-axis and MHC class II on the y-axis. Negative cells are in the boxed area. Note that the majority of the LDC is ED2+ and approximately half MHC class II+. The latter population is also the strongest positive for ED2 (for quantitative data, see F). (B) Dot plot of thymus LDC with OX62 expression on the x-axis and MHC class II on the y-axis. Note that only a minority of LDC is intermediately positive for OX62 (for quantitative data, see F). (C) Immune cytology of thymus LDC with ED2 (red) and MHC class II (blue). Original magnification, x1000; no counterstaining. It can be seen clearly that the majority of cells is ED2+, and that about half of them stain double for MHC class II (particularly the large cells). (D) Immune cytology of thymus LDC with ED2 (red) and OX62 (blue). Original magnification, x1000. A weaker staining was used for ED2 as compared with C by shorter development of the staining step, as OX62 staining appeared rather faint; no counterstaining. It shows that part of the ED2+ cells is also faintly OX62+. (E) Immune cytology of thymus LDC with MHC class II (blue). Original magnification, x1000. Overstaining was used by longer development of the staining step to visualize the long, cellular protrusions (branches/dendrites) clearly; no counterstaining. (F) Quantification of marker-positive LDC in flow cytometric analysis. MFI, Mean fluorescence intensity.

 
Using the DC-specific marker OX62, only 31% (SD 9%, n=7) of the thymus LDC were positive in flow cytometric analysis with a dim staining manner (Fig. 1B and 1F) , and in immunocytology, OX62 was found to be stained weakly in 20–30% of the cells (Fig. 1D) . In double immunocytological staining, it appeared that all OX62+ cells were also positive for ED2, a marker that showed a much stronger staining pattern (Fig. 1D and 1F) .

About half (48%±13, n=7) of the total thymus LDC was positive for MHC class II. This also applied to the ED2+ LDC (Fig. 1A 1C and 1F) . It must be noted that the MHC class II+ED2+ cells were the strongest positive for ED2 (Fig. 1A) . Of the OX62+ cells, approximately two-thirds was positive for MHC class II (Fig. 1B and 1F) . In immunocytology, many of the MHC class II+ cells showed strong branched/dendritic morphology (Fig. 1E) . Only small minorities of the thymus LDC were positive for the macrophage markers ED1 and ED3 (Fig. 1F) . The CD80, CD86, and CD40 expression of the LDC was, respectively, 36 ± 11, 41 ± 13, and 42 ± 7% (mean±SD, n=7).

It is also important to note that the LDC fractions did not contain any cells with an epithelial morphology.

As the LDC were branched/DC and clearly positive for ED2/CD163 (and only in minority for OX62), we were of the opinion that the LDC better fit the phenotypic and morphological description of thymus branched cortical macrophages [26 ] than the medullary and cortico-medullary thymus DC, as described previously [18 , 19 ]. In addition, therefore, we stained thymus sections for ED2 and OX62 to verify the in situ phenotype of the cells. Indeed, a hallmark of the branched cortical macrophages was their strong ED2 positivity, and only few ED2+ cells were found in the medulla of the thymus (Fig. 2A ). OX62+ cells were found predominantly in the medulla and particular, at the cortico-medullary junction (Fig. 2B) , and there was some faint positivity of cells in the cortical area. Based on these phenotypic characteristics, we consider our isolated LDC as representing branched cortical macrophages.


Figure 2
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  		Figure 2. (A) Immunohistochemistry of a F344.+/+ rat thymus stained for ED2 (original magnification, x150). The ED2+ cells in the cortex (i.e., the branched cortical macrophages) are clearly visible. (B) Immunohistochemistry of a F344.+/+ rat thymus stained for OX62 (original magnification, x150). The positivity for OX62 of the cortico-medullary and medullary DC can be seen clearly.

 
In addition, when we compared the phenotype of the isolated thymus LDC with classical DC, i.e., spleen LDC and BM precursor-derived DC using flow cytometric analysis, it is clear that the latter two populations contained many more classical DC than our thymus LDC population, i.e., of cells brightly and intermediately positive for OX62 and clearly positive for MHC class II (Fig. 3 ).


Figure 3
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  		Figure 3. Dot plots of thymus LDC (A), spleen LDC (B), and cultured BM precursor-derived DC (C) with OX62 on the x-axis and MHC class II on the y-axis. Percentages of cells in each quadrant are indicated in the dot plots.

 
The T cell stimulatory capacity of thymus LDC
With regard to the functional capability of the thymus LDC, we compared the T cell stimulatory capacity of thymus LDC with that of spleen LDC and BM precursor-derived DC. Figure 4A and 4B , shows that the thymus LDC were potent stimulators of allogeneic T cells but reached half the potency of spleen LDC. Thymus LDC and spleen LDC were more potent than BM precursor-derived DC. The latter were in fact quite weak T cell stimulators. This is in accord with their relative immature character. We have reported previously that stimulating such BM precursor-derived DC with LPS to mature the cells further does up-regulate their MHC class II expression and their T cell stimulatory capacity to levels found with spleen LDC [24 ].


Figure 4
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  		Figure 4. The T cell stimulatory capacity of thymus LDC (circles), spleen LDC (squares), and BM-derived DC (BMDC; triangles). (•) Thymus LDC of lymphopenic rats. The T cell stimulatory capacity was measured using Wistar T cells as responder cells in allogeneic MLR for F344 rats (A) and in syngeneic MLR for BB rats (B) and expressed as [3H]TdR incorporation (cpm) in stimulated T cells. Means ± SEM are given (n=4). *, Significances (P<0.05) are given, meaning differences between thymus LDC of lymphopenic versus nonlymphopenic rats.

 
Thymus LDC of BB-DP and F344.lyp/lyp rats show a reduced capability to stimulate T cells yet have a normal phenotype except for a lower expression of CD86
T cell stimulatory capacities of thymus LDC of lyp/lyp and nonlymphopenic rats were tested in MLR using Wistar T cells as responder cells, representing an allogeneic MLR for F344 rats (Fig. 4A) and a syngeneic MLR for BB rats (Fig. 4B) . As can be seen, the T cell stimulatory capacity of thymus LDC of lyp/lyp rats was significantly lower than that of nonlymphopenic rats, being it BB-DP or Fischer lyp/lyp rats [ratio APC:T cell, 1:5 (cpm); mean±SEM; non-lyp BB: 32,923±19,240, BB.lyp/lyp: 16,915±9905 (n=4; P<0.05); non-lyp F344: 33,091±9462, F344.lyp/lyp: 15,432±5374 (n=4; P<0.05); Mann-Whitney paired analyses was performed, compare open and closed circles].

Despite this clear difference in T cell stimulatory capacity between thymus LDC of lyp/lyp and nonlymphopenic rats, the phenotype of the LDC fractions was not different, and equal numbers of ED2+, OX62+, and MHC class II+ cells were found in the LDC fractions of lyp/lyp and nonlymphopenic rats. With regard to the costimulatory molecules, there was a difference in the expression of CD86 between lyp/lyp and nonlymphopenic rats: 25 ± 6% (n=7) thymus LDC of lyp/lyp rats were positive for CD86 against 33 ± 9% (n=9) of LDC of nonlymphopenic rats (P<0.05). The expression of CD80 and CD40 was equal.

Thymus LDC of lyp/lyp rats show a lower capability to rescue double-positive thymocytes from apoptosis as compared with nonlymphopenic rats
Thymus LDC of lyp/lyp and nonlymphopenic BB and Fischer rats were cocultured with double-positive thymocytes of lyp/lyp and nonlymphopenic animals. Thymus LDC of nonlymphopenic animals were able to rescue thymocytes from cell death (Fig. 5A and 5D ), further supporting the view that the thymus LDC represent branched cortical macrophages, as these cells are thought to be involved in positive selection [26 , 27 ].


Figure 5
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  		Figure 5. The thymocyte rescuing capability of rat thymus LDC. After coculture of thymus LDC with nonlymphopenic thymocytes, cell death of double-positive thymocytes was assessed via 7-AAD staining in CD4+CD8+ cells. Positivity for 7-AAD is given on the horizontal axis of the histograms and represents cell death. The upper rows depict experiments in F344 rats and the lower rows in BB rats. (C, F) Representative histograms of thymocytes cultured in the absence of thymus LDC. (A, D) Representative histograms of thymocytes cultured in the presence of thymus LDC of nonlymphopenic rats. (B, E) Representative histograms of thymocytes cultured in the presence of thymus LDC of lymphopenic rats.

 
Thymus LDC of lyp/lyp animals hardly rescued thymocytes from apoptosis (Fig. 5B and 5E) , indicating that such LDC are functionally defective. This defect was independent of the thymocyte population used, being similar for thymocytes of lyp/lyp and nonlymphopenic animals (not shown). Also, these two thymocyte populations showed similar survival kinetics when cultured in isolation; i.e., both showed a high level of apoptosis (over 95%) in the absence of rescuing APC.

We also analyzed by flow cytometry (in two experiments) the thymocytes exposed to the thymus LDC of lymphopenic and nonlymphopenic BB and Fischer rats and paid in particular attention to the rescue of the population of CD4+ART2+ T cells. The exposure of the thymocytes to thymus LDC of lymphopenic strains (BB-DP and Fischer.lyp/lyp) resulted in a lower percentage of CD4+ART2+ T cells in the stimulated thymocyte population as compared with percentages found in thymocytes stimulated by thymus LDC of nonlymphopenic BB and Fischer rats (BB-DR and Fischer.+/+): i.e., 21% for BB-DP versus 35% for BB-DR and 13% for Fischer.lyp/lyp versus 23% for Fischer.+/+. This shows that the cortical branched macrophages of lyp/lyp rats are also less capable of rescuing T cell populations of the thymus, which contain an important fraction of the special Treg cells of the rat.

Ian5/Gimap5/lyp is highly expressed in thymus LDC and is decreased in thymus LDC of lymphophenic rats
We studied the expression of Ian5 transcripts in the thymus LDC, spleen LDC, and BM precursor-derived DC and spleen macrophages. We found that the thymus LDC expressed high levels of Ian5 mRNA comparable with the expression levels in thymus T cells (Fig. 6 ). Spleen macrophages also showed high expression levels of Ian5 mRNA. Classical DC (spleen LDC and BM precursor-derived DC) expressed low levels of Ian5 mRNA (Fig. 6) . These data further support the idea that thymus LDC are macrophages rather than DC. It is interesting that thymus LDC of lymphopenic (lyp/lyp) rats showed a decreased Ian5 mRNA expression (45% as compared with thymus LDC of +/+ rats), and thymus LDC of lyp/+ rats showed an intermediate Ian5 mRNA expression (74% as compared with thymus LDC of +/+ rats; Fig. 6 ). Similar observations of reduced Ian5 mRNA have been made for lyp/lyp T cells [10 , 11 ].


Figure 6
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  		Figure 6. The expression of Ian5 mRNA in thymus LDC of F344.+/+, lyp/+, and lyp/lyp rats and in spleen macrophages, spleen LCD, BM precursor-derived DC, and thymocytes of F344.+/+ rats. The Ian5 expression is normalized against cyclophilin expression.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Within the thymus, nonlymphoid cells play an important role in the generation of thymocytes. Sessile epithelial and mobile accessory cells are the two prominent groups of cells within this nonlymphoid compartment. The latter appear as two distinct cell types: the thymus DC and the thymus macrophages [28 ]. The thymus DC are predominantly located in the medulla, express characteristic markers of DC (such as OX62 in the rat; see this report), and play a role in the negative selection of thymocytes. With regard to the thymus macrophages, two different subpopulations can be identified: large, rounded macrophages (in the rat positive for ED1) with a high phagocytic activity present in the cortex and medulla (and probably playing a role in the removal of apoptotic thymocytes) and nonphagocytic, large branched cortical macrophages [29 ]. In rats, these latter cells are characterized by their dendritic morphology and labeling with mAb to ED2/CD163 [25 ]. The branched cortical macrophages have a slow turnover (in contrast to the other thymus macrophage population and the thymus DC) and probably originate from BM precursors (although an intrathymic precursor has also been suggested for part of the cells) [30 ]. Functionally, the cells have intimate contact with the cortical thymocytes and are capable of forming cell complexes with double-positive thymocytes in vitro. In addition, the cells are largely positive for MHC class II molecules, and it has therefore been suggested that branched cortical macrophages play a role in thymocyte maturation and/or differentiation in the cortex [30 ].

Our data show that the thymus LDC, which we isolated, fitted best the phenotype and suggested function of the rat branched cortical macrophages: The cells were predominantly positive for ED2 (and only for a proportion, weakly positive for the DC marker OX62) and had a branched/dendritic morphology, half the cells were positive for MHC class II, and the cells were excellent T cell stimulators and rescued double-positive thymocytes from apoptosis.

Thymus LDC have been isolated from the rat thymus before by the group of Ardavin and by Ilic et al. [18 , 19 ]. In the reports of these investigators, the LDC were considered to be the thymus medullary DC on the basis of their branched morphology, their MHC class II positivity, and excellent T cell stimulatory capacity. We found these qualities as well. However, we also found the cells hardly positive for OX62, and OX62 proved to be a good marker for rat medullary thymus DC in situ. We are therefore of the opinion that thymus LDC fractions predominantly contain ED2+ branched cortical macrophages and not classical DC (Banuls et al. [18 ] also noted some ED2 positivity of the thymus LDC). Conversely, we must note that Banuls et al. [18 ] and Ilic et al. [19 ] used slightly different isolation techniques to obtain thymus LDC, and this might have resulted in a predominant yield of medullary thymus DC. Banuls et al. [18 ] isolated thymus DC predominantly from the thymus LDC by using two consecutive adherence steps, and Ilic et al. [19 ] obtained mainly thymus DC by culturing thymus LDC for 3 days with conditioned medium derived from medullary thymic epithelial cells, which contained the cytokines IL-1, IL-6, TNF-{alpha}, and IL-2.

A second novel observation we made was that the thymus LDC populations of the BB-DP and F344.lyp/lyp rat were deficient in function as compared with the thymus LDC of nonlymphopenic BB-DR and F344 rats: the cells had a reduced T cell stimulatory capacity and, more importantly for branched cortical macrophages, a reduced capability to rescue double-positive thymocytes from apoptosis. This phenomenon of a reduced capability to rescue double-positive thymocytes from apoptosis is of interest, as it could explain at least part of the severe lymphopenia characteristic of these rats. Classically, the lyp is thought to be a result of a shortened lifespan of recently emigrated thymocytes because of a high, "spontaneous" apoptosis of such cells. There are, however, previous reports that BB-DP thymocytes can at least be rescued partially from such a shortened lifespan. Ramanathan et al. [31 ] showed that an early exposition to antigen could partially rescue recent thymic emigrants of the BB-DP rat from programmed cell death. In particular, thymus branched cortical macrophages would be capable of delivering such early antigenic signals to developing thymocytes.

Is this inability of branched cortical macrophages in the BB-DP rat linked to the proneness for autoimmunity? As our LDC fraction did not contain epithelial cells, we hold the APC as responsible for the rescue of the double-positive thymocytes. The mechanisms via which thymic APC induce tolerance are presently not known. Next to the MHC class II+ thymus epithelial cells, the thymus APC shape the T cell repertoire via positive selection, including that of Treg cells and via negative selection deleting strongly autoreactive T cells.

Several reports of Georgiou et al. [15 16 17 ] support the idea that appropriately functioning APC in the thymus are able to prevent type I diabetes by partially restoring T cell abnormalities. These investigators showed that the transplantation of a nonlymphopenic thymus led to the restoration of T cell-proliferative function in the BB-DP rat, significantly reduced the incidence of insulitis, and prevented the development of diabetes. It appeared from their experiments that a defect in BM-derived thymus APC contributed to an abnormal maturation of BB-DP T lymphocytes, which in turn, predisposed the animals to autoimmune insulitis, as the transplantation of "healthy" thymus APC restored the T cell population and prevented disease. In the NOD mouse, Georgiou et al. [32 , 33 ] showed that the neonatal transfer of healthy thymus macrophages also prevented disease. It is thus tempting to speculate, in view of our data, that the lymphopenia-restoring and diabetes-preventing APC of the transplant experiments of Georgiou et al. [32 , 33 ] are able to act as the here-described thymus cortical branched macrophages rescuing double-positive thymocytes from apoptosis. As these macrophages also rescue ART2+ T cells (see this report), which harbor thymus-derived Treg cells, they might, in this way, contribute to prevention of disease.

With regard to the expression of relevant autoantigens in the thymus (e.g., the insulin peptide family), Kecha-Kamoun et al. [34 ] reported that thymic macrophages express, in particular, insulin growth factor 2 (IGF-1), and thymus epithelial cells express IGF-2, and thymic DC and medullary epithelial cells express insulin. The investigators showed that in the BB-DP rat model, IGF-2 expression was defective in 11 of 15 thymuses in close concordance with the diabetes incidence in their rat strain (and IGF-1 and insulin expression were intact). Hence, their experiments do not point to a defective function of thymus macrophages in the BB-DP rat at least at the level of autoantigen expression.

Which mechanisms might be responsible for the decreased function of branched thymus cortical macrophages of BB-DP and the Fischer lyp/lyp rats? It is interesting that the thymus LDC of lyp/lyp rats showed a reduced expression of Ian5 mRNA as compared with thymus LDC of +/+ rats, and lyp/+ rats showed intermediate levels. A similar expression pattern exists in lyp/lyp, lyp/+, and +/+ thymocytes in the BB rat model, and the severely reduced levels of Ian5 in lyp/lyp thymocytes lead to an enhanced apoptosis. We did not study whether the lyp/lyp LDC had an increased apoptosis rate, but it is interesting that Van Rees and Dijkstra [35 ] have described an in situ reduction in the number of thymus cortical ED2+ macrophages in BB-DP rats. Obviously, we need to test the exact yield and apoptosis rate of LDC isolated from BB-DP and F344.lyp/lyp rats in future experiments.

In conclusion, we found that nonadherent LDC isolated from the thymus represent ED2+ branched cortical macrophages and that in the BB-DP and F344.lyp/lyp rat, such cells had a reduced Ian5 expression, T cell stimulatory capacity, and a strongly diminished capability to rescue thymocytes from apoptosis, including that of ART2+ T cells. It is likely that these defects, in this important macrophage population of the thymus, are at least in part responsible for the severe T cell lymphocytopenia and the lack of Treg cells in the periphery of lyp/lyp gene-carrying rats.


   ACKNOWLEDGEMENTS
 
The Netherlands Organization for Scientific Research (grant no. 903-40-193) and the National Institutes of Health (AI42380, DK 17047) supported this work. We gratefully acknowledge Mr. H. de Wit for immunohistochemical assistance, Mr. H. Dronk for care of the animals, and Mr. T. van Os for photographic assistance.

Received April 6, 2007; revised May 10, 2007; accepted May 10, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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