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The Journal of Immunology, 2005, 175: 1965-1973.
Copyright © 2005 by The American Association of Immunologists

The Role of Fas in the Immune System Biology of IL-2R{alpha} Knockout Mice: Interplay among Regulatory T Cells, Inflammation, Hemopoiesis, and Apoptosis

Rahul Sharma, Harini Bagavant, Wael N. Jarjour, Sun-Sang J. Sung and Shyr-Te Ju1

Division of Rheumatology and Immunology, Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Introducing lpr mutation prevents early mortality associated with IL-2R{alpha} knockout (KO) mice, prompting us to determine the role of Fas in the immune system biology of IL-2R{alpha} KO mice. Consistent with a defect in CD4+CD25+ regulatory T (Treg) cell expression, spontaneous lymphocyte activation in lymphoid organs was observed in 6-wk-old mice. In 16- to 22-wk-old mice, infiltration of leukocytes was observed in bone marrow, colon, lung, pancreas, lacrimal gland, and salivary gland, but not in heart, thyroid, liver, stomach, small intestine, ovary, and kidney. In the lymphocytes-infiltrated bone marrow, B cell lymphopoiesis was blocked at pro-B to pre-B/immature B stage, culminating in an age-dependent B cell loss in the periphery. These phenotypes were also observed in IL-2R{alpha} KO mice bearing the lpr mutation (DM mice), indicating Treg cell function and the phenotypes attributed directly to Treg cell abnormality are largely Fas-independent. However, anemia and body weight loss were partially prevented, tissue cell apoptosis was inhibited, and lifespan was improved in the DM mice, demonstrating Fas-dependent elements in these processes. Our age-dependent, lifelong analysis of IL-2R{alpha} KO and DM mice supports a CD4+CD25+ Treg cell-based mechanism for the abnormal immune system biology observed in IL-2R{alpha} KO mice and provides a global view of the interplays among Treg cells, multiorgan inflammation, hemopoiesis, and apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The IL-2/IL-2R signaling pathway plays a critical role in the immune system (reviewed in Refs. 1, 2, 3). It is a powerful signal for lymphocyte proliferation and a critical factor for the development and expansion of the CD4+CD25+ regulatory T (Treg)2 cells that down-regulate T cell proliferation/activation. A characteristic phenotype of Treg cell absence is tissue inflammation (4, 5, 6, 7). Targeted mutation of IL-2, IL-2R{alpha} (CD25), or IL-2R{beta} (CD122) gene results in a similar phenotype characterized not only by tissue inflammation as exemplified by ulcerative colitis but also by severe anemia, body weight loss, and other immune abnormalities such as B cell deletion reported in old IL-2 knockout (KO) mice and IL-2R{beta} KO mice (8, 9, 10, 11, 12, 13, 14, 15). A consequence associated with these abnormalities is early mortality, which prevents a thorough analysis of the immune system defect and immune regulation in these mice, especially in adult and older mice.

The FasL (CD178)/Fas (CD95)-mediated apoptosis pathway is critical to the maintenance of peripheral tolerance, most notably in systemic autoimmune disease such as the lupus-like autoimmune disease in lpr and gld mice (16, 17). In addition, FasL on infiltrating lymphocytes of the inflamed tissues have been shown to induce apoptosis of tissue cells (18, 19, 20, 21). We recently have shown that introducing the lpr mutation into IL-2 KO mice prolongs their lifespan (21). This is attributed at least in part to the prevention of anemia and in part to the prevention of the FasL-induced apoptosis of colon epithelial cells. However, whether Fas-mediated signaling pathways are involved in a specific phenotype such as B cell deletion, tissue inflammation, tissue cell death (other than colon) was not determined (21).

It is likely that a common mechanism is responsible for many of the phenotypes shared among IL-2 KO, IL-2R{beta} KO, and IL-2R{alpha} KO mice. We propose a hypothesis based on a current concept of Treg cell function to explain the shared phenotypes among these mouse strains. We hypothesize that the absence of Treg cells allows CD4+CD25– T cells (including autoimmune T cells) to be polyclonally activated. Activated autoimmune T cells infiltrate specific target organs where they interfere with organ functions, leading to various phenotypes. In addition, autoimmune T cells that target bone marrow are induced, and bone marrow is highly sensitive to these cells. T cell infiltration into bone marrow suppresses bone marrow function, leading to progressive hemopoiesis-based abnormalities. We choose to study IL-2R{alpha} KO mice because T cell infiltration into bone marrow, B cell deletion, and inflammation in multiple organs were not described (13). Also, we introduced lpr mutation into IL-2R{alpha} KO mice (referred to as DM mice) to determine whether a specific phenotype is dependent on Treg cells or Fas expression. This is significant because Fas mutation prolonged the lifespan of IL-2R{alpha} KO mice. Fas mutation did not prevent inflammation in multiple organs. However, it affected the pathology of the inflamed lung and colon. Like IL-2 KO and IL-2R{beta} KO mice, we also observed T cell infiltration into bone marrow where erythropoiesis and B cell lymphopoiesis were impaired, culminating a progressive, age-dependent anemia and peripheral B cell deletion. Collectively, our study provides a mechanism-based description of the immune system biology throughout the lifespan of IL-2R{alpha} KO mice by characterizing their specific immune phenotypes and by defining the roles of CD4+CD25+ Treg cells and Fas in these phenotypes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Breeding and genotyping

C57BL/6.Il2R{alpha}+/– mice and B6.MRL-Faslpr/J (B6.lpr or lpr+/+) mice were obtained from The Jackson Laboratory. Because Il2R{alpha}–/– (IL-2R{alpha} KO) mice are sterile, Il2R{alpha}+/– mice were first bred with lpr+/+ mice to obtain Il2R{alpha}+/–lpr+/– F1 offspring. F1 mice were intercrossed to obtain Il2R{alpha}–/–lpr+/+ and Il2R{alpha}+/–lpr+/+ mice. We then intercrossed Il2R{alpha}+/–lpr+/+ mice to increase the frequency of Il2R{alpha}–/–lpr+/+ offspring. Tail DNA preparations of 4-wk-old mice were used for genotyping by PCR. The primers and condition used for fas PCR analysis have been described (21). The Il2R{alpha} gene was analyzed by PCR using the primer sequences and reaction conditions from The Jackson Laboratory website (<www.jax.org>). PCR products were determined by their sizes as follows: 218 bp for fas mutant of lpr mice, 182 bp for the normal fas, 280 bp for the Il2R{alpha} mutant allele, and 146 bp for the normal Il2R{alpha} allele. DNA samples from C57BL/6J (B6), IL-2R{alpha} KO, and B6.lpr mice were used as controls. Mice were housed at the University of Virginia animal facility, and experiments were conducted following the protocol approved by the Institutional Animal Care and Use Committee.

General examination of mice

Mice were examined twice weekly for mortality. Starting at 4 wk of age, mice were weighed and blood samples were collected every 2 wk. Ten microliters of blood samples were collected and immediately diluted in 90 µl of cold PBS, pH 7.2. Aliquots were used to determine hemoglobin levels and cell-free samples were collected by centrifugation and used for the determination of IgG levels using the mouse IgG ELISA kit (Bethyl Laboratories).

Determination of hemoglobin level

Blood samples were diluted 100-fold with dilution buffer (3 mM potassium ferricyanide, 1.5 mM potassium cyanide, 5 mM sodium borate, 0.1% Nonidet P-40) in which RBC were lysed and the released hemoglobin was converted to cyanomethemoglobin. The relative hemoglobin concentrations were determined photometrically at 546 nm.

Immunohistochemical and immunofluorescence staining

Sections of paraffin-embedded tissues were stained with H&E. Tissues examined are lungs, thyroids, lacrimal glands, salivary glands, hearts, stomachs, livers, pancreas, kidneys, small intestines, colons, and ovaries. Apoptosis was determined using a TUNEL assay kit (Apoptag Plus Peroxidase In Situ Apoptosis Detection kit; Serologicals). We followed the manufacturer’s protocol and included the positive control provided by the manufacturer. Background staining was determined by incubation with buffer in the absence of TdT. Slides were counterstained with methylene blue followed by graded alcohol dehydration and mounting. TUNEL+ cells developed a characteristic brown/black color in the nuclear region.

Confocal microscopy

Frozen sections of lymph nodes were stained for 30 min with PE-conjugated anti-B220 (RA3-6B2) and hamster anti-CD3 (145-2C11) (BD Biosciences). After washing, slides were incubated for 30 min with Alexa 647-conjugated goat anti-hamster Ab (Molecular Probes). For a negative control, we used rat IgG and hamster IgG (both from BD Biosciences) in place of anti-B220 mAb and anti-CD3 mAb, respectively. The slides were examined using a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss).

Flow cytometric analysis

Lymphocytes in blood, lymph nodes, spleen, bone marrow, and thymus were analyzed. Single cell suspensions of samples were treated with ammonium chloride to remove erythrocytes, washed, and then stained for various cell surface markers. Cells (106) were suspended in 100 µl of PBS containing 4% BSA and incubated with 1 µg of various fluorescent Abs for 30 min at 4°C. FITC- and/or PE- conjugated anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-Thy-1.2 (30H12), anti-B220 (RA3-6B2), anti-CD43 (S7), anti-Fas (Jo-2), and anti-CD69 (H1.2F3) mAb were obtained from BD Biosciences. FITC conjugated goat anti-mouse IgM was purchased from Southern Biotechnology Associates. At least 104 stained cells were analyzed using a FACScan (BD Biosciences) equipped with CellQuest (BD Biosciences). Post acquisition analyses were conducted using FlowJo software (Tree Star).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Fas mutation prevents early mortality and prolongs the lifespan of IL-2R{alpha} KO mice

We introduced the fas mutant gene of B6.lpr mice into IL-2R{alpha} KO mice by breeding. The genotypes of progeny were identified by PCR on tail DNA samples (Fig. 1A). The cell surface phenotypes were demonstrated by staining lymphocytes with FITC-anti-Fas mAb for Fas expression and FITC-anti-CD4 plus PE-anti-CD25 mAb for Treg cells (Fig. 1, B and C). Mice were observed weekly for general appearance and mortality. In addition, body weight gain/loss and the severity of anemia were determined. The compiled data are shown in Fig. 2. Mice bearing both Fas and IL-2R{alpha}-targeted mutations (DM mice) survived significantly longer than IL-2R{alpha} KO mice but did not reach to the life span of B6.lpr and B6 mice (Fig. 2A). Severe anemia as defined by ≤50% of control B6 hemoglobin level was observed in 10-wk- but not 4-wk-old IL-2R{alpha} KO and DM mice (Fig. 2B). Interestingly, the protection of anemia by lpr mutation in DM mice is moderate for males and unremarkable for females (Fig. 2B). The reason for the differential protection is unclear. A moderate protection of body weight loss as compared with IL-2R{alpha} KO mice was observed in male DM mice but not in the female DM mice (Fig. 2C). Still, Fas mutation prolonged the lifespan of female mice as well as male mice. The partial and marginal improvement of body weight gain and the weak prevention of anemia agree with the inability of these mice to reach the lifespan of B6.lpr and B6 mice.



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  		FIGURE 1. Genotyping and phenotyping IL-2R{alpha} KO and DM mice. A, The PCR product sizes are 218 and 182 bp for the lpr and wild-type fas allele, respectively. The PCR product sizes for the mutant and wild-type IL-2R{alpha} are 280 and 146 bp, respectively. In this example, lane 1 and lane 8 represent DM mice, lane 3 has the IL-2R{alpha} KO genotype, lane 5 is lpr, and lane 7 is a wild-type mouse. Lane 6 is molecular size standard. B, Lymph node cells of genotyped mice were stained with FITC-anti-Fas mAb for Fas expression and analyzed with flow cytometry (shaded, isotype; thin line, DM; thick line, B6). C, The genotyped DM mouse is negative for CD4+CD25+ Treg cells.

 


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  		FIGURE 2. Fas mutation protects IL-2R{alpha} KO mice from early mortality, anemia, and body weight loss. The DM mice lived longer than IL-2R{alpha} KO mice (A) and had improved blood hemoglobin levels (B, male; C, female) and body weight (D, male; E, female). The number of mice examined are equal to or more than six (n ≥ 6) except for IL-2R{alpha} KO mice (n = 5 for male and n = 3 for female).

 
Spontaneous T cell activation in 6-wk-old IL-2R{alpha} KO and DM mice

We determined whether spontaneous T cell activation occurs as a result of lifelong absence of the CD4+CD25+ Treg cells. As shown in Fig. 3, when compared with age- and sex-matched B6 mice, a significantly higher proportion of the CD4+ T cells in the lymph nodes of 6-wk-old IL-2R{alpha} KO (6 of 12 or 50%) and DM (7 of 18 or 39%) mice expressed the activation marker CD69, whereas in B6 mice the value was 16%. Similarly, increase in CD69 expression was observed for CD8+ T cells. We noted that there was an increase in CD8+ T cells in both IL-2R{alpha} KO and DM mice (data not shown) and the proportion of CD69+ cells within the CD8+ T cell population was somewhat less than the CD69+ cells in the CD4+ T cell population.



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  		FIGURE 3. Spontaneous activation of T cells in IL-2R{alpha} KO and DM mice. Lymph node cells from 6-wk-old female B6, IL-2R{alpha} KO, and DM mice were stained with anti-CD4 mAb plus anti-CD69 mAb (upper panels) and anti-CD8 mAb plus anti-CD69 mAb (lower panels). Please note that the numbers in the quadrants are percentages of the population of total lymphocytes.

 
Fas mutation did not prevent tissue inflammation

We determined whether IL-2R{alpha} KO mice (3–4 mo old) develop inflammatory response in multiple tissues/organs following the spontaneous T cell activation. Various tissues were collected from B6, B6.lpr, IL-2R{alpha} KO, and DM mice and the stained tissue slides were examined. As shown in Fig. 4, IL-2R{alpha} KO and DM mice showed histopathological changes in lungs, colon, pancreas, and lacrimal glands in comparison to the counterparts of B6 and B6.lpr (data not shown) mice. Both IL-2R{alpha} KO and DM mice developed lung inflammation. The severity of inflammation was greater in the IL-2R{alpha} KO mice compared with DM mice. Both showed cellular infiltrates surrounding the bronchiolar lumen extending into the lung parenchyma. However, there were differences in the nature of the infiltrating cells. Lymphocytes were the major infiltrating cells in all six DM mice examined. In three of the six IL-2R{alpha} KO mice examined, lymphocytes were also the major cell type in the affected regions. However, strong infiltration of both neutrophils and lymphocytes were observed in the other three mice. In some areas, the neutrophil-containing exudates formed a plug in the bronchiolar lumen along with hypertrophy of the lining epithelium. The reason why neutrophil infiltration was observed in the lung of some but not all IL-2R{alpha} KO mice is not clear at present but its presence is consistent with the idea that FasL is a potent chemotactic factor for neutrophils (22).



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  		FIGURE 4. Histological examination of tissues from B6, IL-2R{alpha} KO, and DM mice. Various tissues were fixed in 10% neutral buffered formalin, paraffin embedded, and sectioned, and 4-µm sections were stained with H&E. Lung (A–C), colon (D–F), pancreas (G–I), lacrimal glands (J–L), and thyroid (M–O) are shown. Lymphocytic infiltration (ly) seen in IL-2R{alpha} KO and DM mice is indicated. IL-2R{alpha} KO mice have a strong neutrophil infiltration (arrows) in the lungs and bronchiolar lumen exudate (B). Arrow in inset in E shows a multinucleated giant cell seen in the colonic mucosa of IL-2R{alpha} KO mice. Photomicrographs were taken at x200 for lung (A–C) and colon (D–F). All other photomicrographs were taken at x100 magnification. Little or no infiltration was observed in tissues of control B6 mouse.

 
The colonic mucosa in both IL-2R{alpha} KO and DM mice showed significant thickening and hypertrophy compared with control B6 mice (Fig. 4). Lymphocytic infiltration in the lamina propria was observed in both groups but was more prominent in the DM mice. Multinucleated giant cells were also observed in the colonic mucosa of IL-2R{alpha} KO mice. The submucosa and muscular layers appeared normal.

Changes in the pancreas and lacrimal glands were characterized by extensive lymphocytic infiltration in the periductal regions. There was little involvement of the exocrine glands. In the pancreas, infiltration did not appear to invade the pancreatic islets or cause islet cell destruction. These mice did not develop diabetes mellitus as determined by urinary glucose level (data not shown). Similar periductal infiltration was seen in salivary glands of both IL-2R{alpha} KO and DM mice. Inflammation in thyroid, liver, stomach, small intestine, ovary, kidney, and heart was either very mild or not observed (data not shown). No pathological changes were apparent in tissues from age-matched control B6 and B6.lpr mice. These data suggest that inflammation in multiple tissues/organs is due to the lack of CD4+CD25+ Treg cells in both IL-2R{alpha} KO and DM mice. In addition, Fas deficiency in DM mice influences the qualitative nature (infiltrating cell type) and extent of inflammation (colon in DM mice) of the inflammatory response.

Fas mutation inhibited apoptosis of tissue cells in the inflamed lung and colon

To determine whether tissue cells in the severely inflamed sites were killed by Fas/FasL-mediated apoptosis, we used the TUNEL assay to determine the cell types and the number of apoptotic cells in the lung and colon of IL-2R{alpha} KO and DM mice. A representative experiment is shown in Fig. 5. Few apoptotic cells were observed in the lung of a B6 mouse. Many apoptotic cells were observed in the lung of the 16-wk-old IL-2R{alpha} KO mouse. Although many of them were leukocytes, apoptotic tissue cells were evident (inset and right panels). In the lung of an age-matched DM mouse, the number of apoptotic leukocytes was significantly reduced in comparison with IL-2R{alpha} KO mouse. In addition, apoptotic tissue cells were rarely observed (inset and right panels). Similarly, a higher number of apoptotic cells were detected in the inflamed colon of IL-2R{alpha} KO mice (Fig. 5). Both apoptotic cells and tissue damage were less evident in the inflamed colon of DM mouse than IL-2R{alpha} KO mouse. These observations suggest that FasL is induced in IL-2R{alpha} KO mice and that Fas-mediated apoptosis plays a major role in cell death during the inflammatory response in IL-2R{alpha} KO mice.



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  		FIGURE 5. Fas mutation protects cell death in the inflamed lung and colon of IL-2R{alpha} KO mice. Sections of lung (top panels) and colon (bottom panels) were stained for apoptotic cells by TUNEL assay. TUNEL+ cells as indicated by dark brown color were more frequent in the IL-2R{alpha} KO mice than DM mice (x100). The inset captured at x400 magnification shows the apoptotic cell types. Both apoptotic leukocytes (arrows) and apoptotic tissue cells (arrowheads) were observed. (Ly, lymphocyte; N, neutrophil). The bar graphs (right panels) show the numbers of TUNEL+ tissue cells, TUNEL+ lymphocytes, and total apoptotic cells in the inflamed tissues. A total of five randomly selected fields were counted for each sample at a magnification of x200. The tissue apoptotic cells were distinguished from apoptotic lymphocytes on the basis of the size of the nucleus and surrounding cytoplasm.

 
IL-2R{alpha} KO mice display Fas-independent, age-dependent deletion of B cells

Blockade of B cell differentiation in bone marrow. Previous studies of IL-2 KO and IL-2R{beta} KO mice have shown that B cell lymphopoiesis was severely blocked at the pro-B to pre-B/immature B cell stage as a result of infiltration of lymphocytes and leukocytes, respectively (12, 14). We also observed a strong infiltration of T cells in the bone marrow of IL-2R{alpha} KO and DM mice (see Table III). Therefore, we examined B cell lymphopoiesis in the bone marrow of both the IL-2R{alpha} KO (16 wk old) and DM (22 wk old) mice using anti-B220, anti-IgM, and anti-CD43 mAb to determine the B cell subpopulations at various differentiation stages in the bone marrow. A representative experiment is shown in Fig. 6. The percentages of B220+IgM– pre-B/immature B cells (from 6.13% in B6 to 0.36% in IL-2R{alpha} KO and 1.03% in DM mice) and B220+IgM+ mature B cells (from 3.01% in B6 to 0.33% in IL-2R{alpha} KO and 0.58% in DM mice) were greatly reduced in the bone marrow of IL-2R{alpha} KO and DM mice, whereas the percentage of B220+CD43+ pro-B/small pre-B cells was not significantly different from a B6 control (4.47% in B6 vs 3.69% in IL-2R{alpha} KO and 4.75% in DM mice). The data indicate that bone marrow B cell differentiation is blocked at the pro-B to pre-B/immature B cells stage and this phenotype is mostly Fas-independent. It is important to note that the total number of bone marrow cell count (after depletion of erythrocytes) was comparable among these mice (data not shown), indicating that the output of naive B cells into the periphery is greatly reduced in the old IL-2R{alpha} KO and DM mice as a consequence of this blockade.



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  		FIGURE 6. B cell development arrest in the bone marrow of IL-2R{alpha} KO mice is Fas-independent. Bone marrow cells from B6, IL-2R{alpha} KO, and DM mice were stained with PE-anti-B220 mAb and FITC-anti-CD43 mAb. B cell development in bone marrow was affected at pre-B/immature B cell stage. Both B220highIgM+ (mature B cells) and B220intIgM– (immature B cells) cells were reduced in IL-2R{alpha} KO and DM mice (top row). The B220+CD43– cells (late pre-B, immature B and mature B cells) were affected in both IL-2R{alpha} KO and DM mice, whereas the B220+CD43+ (pro-B and early pre-B) population was not (bottom row). The B6 and DM mice were 22 wk old while the IL-2R{alpha} KO mouse was 16 wk old. A representative of three experiments is shown.

 
Depletion of B cell zone in lymph nodes. We reasoned that a direct consequence of lacking an input of B cells from bone marrow could result in B cell depletion in the periphery. Following the immature B cell differentiation blockade in the bone marrow, the area of B cell zone in the lymph nodes of 14-wk-old mice was proportionally reduced as demonstrated by two-color staining using anti-B220 mAb/anti-CD3 mAb and examined with a confocal microscope (Fig. 7). This reduction is significant considering the fact that the lymph node size of IL-2R{alpha} KO and DM mice was much larger than B6 control. In addition, a more severe B cell zone depletion was observed in 22-wk-old DM mice (data not shown). Interestingly, the remaining B cell zone was infiltrated with T cells. Infiltration of T cells into B cell zone and loss of T/B cell demarcation was also observed in the B6.lpr lymph node as previously reported (23). Consequently, both Treg cell abnormality and Fas mutation could contribute to the T cell infiltration into B cell zone in DM mice.



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  		FIGURE 7. IL-2R{alpha} KO and DM mice display disrupted lymph node structure. Frozen sections of inguinal lymph nodes from B6, B6.lpr, IL-2R{alpha} KO and DM mice were stained for B cells (red) and T cells (blue) using PE-anti-B220 mAb and anti-CD3 mAb followed by Alexa 647-conjugated goat anti-hamster Ab, respectively (top panels) and examined under a confocal microscope. All mice used in this experiment were 14 wk old. Note the lymph node size difference between B6 and mutant mice. The bottom panels focus on the B cell zones demarcated in the top panels. Reduction of B cell zones and infiltration of T cells into the B cell zones were observed for B6.lpr, IL-2R{alpha} KO, and DM mice.

 
Depleting serum IgG caused by early B cell activation in IL-2R{alpha} KO mice and late B cell activation in lpr mice. Early polyclonal lymphocyte activation and the subsequent B cell deletion should affect serum IgG levels. Therefore, the total serum IgG levels of mice at various ages were determined (Fig. 8). The IgG level of B6 mice did not fluctuate much during the entire period of study. As early as 1 mo old, the IgG level of IL-2R{alpha} KO mice was already significantly higher than B6. This level was maintained for a month and then declined for the next 2 mo to a level slightly lower than B6 control. The IgG level of B6.lpr was normal up to 2 mo of age and sharply increased to as high as 350% of the IgG level of B6 mice. The age-dependent pattern of IgG expression level in the DM mice resembles that of the IL-2R{alpha} KO mice but not the B6.lpr mice (Fig. 8). Especially, the increase in IgG level in old B6.lpr mice was not observed in the old DM mice. The serum IgG level in the 22-wk-old DM mice dropped to 30% of the level of B6 mice at 9 mo of age (Fig. 8). The data demonstrated a dominant role of Treg cell abnormality over Fas mutation in regulating serum IgG level. This dominance is consistent with the early lymphocyte activation and the late B cell deletion in the IL-2R{alpha} KO and DM mice.



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  		FIGURE 8. Age-dependent changes in serum IgG levels. Serum samples from individual mice (n = 3) obtained at specific age were pooled. IgG levels were determined by ELISA as described in Materials and Methods. The pattern of IgG levels of DM mice resembles that of IL-2R{alpha} KO mice but not B6.lpr mice. The IL-2R{alpha} KO and DM mice had very high serum IgG levels at a young age that progressively declined with age. Early mortality prevented sample procurement from IL-2R{alpha} KO mice >4 mo old. The data is a representative of three similar experiments.

 
The age-dependent elevation of IgG followed by the dramatic decline in the sera of IL-2R{alpha} KO and DM mice is consistent with the hypothesis that absence of Treg cells in these mice permitted an early T cell-dependent polyclonal activation of B cells, and a subsequent deletion of B cells resulted from T cell infiltration into bone marrow. In addition, the deranged lymphoid architecture in these mice may not allow optimal T/B interaction and this may also contribute to the age-dependent decline in serum IgG level. Finally, the slower rate of decline of the serum IgG level in the DM mice in comparison to IL-2R{alpha} KO mice suggests a small role of Fas mutation in this phenotype by protecting lymphocytes from FasL-mediated apoptosis.

Changes in peripheral B cell numbers.

Because we also observed changes in B cell proportion in the blood samples of IL-2R{alpha} KO and DM mice, the age dependence of this phenotype was determined for individual mice by staining blood samples obtained at various age points with anti-B220 and anti-Thy-1 mAb (Fig. 9). At 4 wk of age, the earliest age analyzed, the percentage of B cells in the lymphocyte population in blood was moderately reduced (70% of B6 control). This value rapidly declined to a plateau of 26% in 10-wk-old mice and 28% in 22-wk-old mice.



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  		FIGURE 9. IL-2R{alpha} KO and DM mice display a progressive, age-dependent reduction of B cells in the blood. Blood samples of individual B6, IL-2R{alpha} KO, and DM mice were obtained at various age points and stained with PE-anti-B220 and FITC-anti-Thy-1.2. IL-2R{alpha} KO mice did not survive for the 22-wk sampling.

 
To determine the actual changes in B cell number and proportion in lymph nodes, spleen, and bone marrow, individual mice were euthanized at specific ages and single cell suspensions were prepared and counted as described in Materials and Methods. Cells were stained with PE-anti-B220 and FITC-anti-Thy-1 mAb, and the proportion was used to determine the total number of T and B cells in each preparation. As shown in Table I, the relative percentages of B cell population in lymph nodes of 4-wk-, 10-wk-, and 22-wk-old DM mice were reduced, respectively, to 82% (24 of 28, p > 0.05), 53% (19 of 36, p < 0.05), and 7% (4 of 58, p < 0.05) of age-matched control mice. The absolute number of B cells in the 4-wk- (5 x 106 in DM vs 3 x 106 in B6) and 10-wk-old (4 x 106 in DM vs 3 x 106 in B6) DM mice was not significantly different from age-matched B6 control. A significant decrease in the absolute B cell number was observed only in 22-wk-old mice (from 4 x 106 of B6 mouse to 1 x 106 of DM mouse), and this agreed well with the reduction of serum IgG level in these mice. This pattern of B cell regulation is also observed in the spleen and in IL-2R{alpha} KO mice (4-, 10-, and 14-wk-old mice were analyzed


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  		Table I. Numbers and percentages of B and T cell in the lymph nodes (LN) and spleens (Sp) of B6, IL-2R{alpha} KO, and DM micea

 
due to early mortality), indicating that B cell deletion in the periphery is mostly Fas-independent. We also observed a progressive, age-dependent increase in Thy-1+ T cells in IL-2R{alpha} KO and DM mice as compared with B6 control (Table I). This increase in Thy-1+ T cells is intrinsic to IL-2R{alpha} KO and is caused by a specific increase in CD8+ T cells in IL-2R{alpha} KO and DM mice (data not shown).

Change in CD4+ T cell numbers in bone marrow

CD4+ T cells from IL-2 KO and IL-2R{beta} KO mice have been shown to transfer into nude mice several major phenotypes (11, 12, 14). Therefore, we determined whether the absolute number of CD4+ T cells was increased in these mice, we used FITC-anti-CD4+ mAb to stain CD4+ T cells in the lymph nodes, spleen, and bone marrow of various mouse strains (Table II). For all lymphoid tissues studied except bone marrow and 22-wk-old DM mice, the total number of CD4+ T cells was comparable to that observed in the age-matched B6 controls, despite the fact that these CD4+ T cells expressed activated phenotype based on CD69 expression (Fig. 3). Although some activated CD4+ T cells must have emigrated out of the lymphoid tissues to the inflamed tissues, this apparent lack of CD4+ T cell expansion in the lymphoid tissues in IL-2R{alpha} KO mice differs from IL-2 KO and IL-2R{beta} KO mice (8, 12, 14, 15). Whether this is due to the intrinsic property of IL-2R{alpha} targeted mutation remains to be determined.


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  		Table II. Comparison of CD4+ T cell numbers obtained from lymph nodes (LN), spleen (Sp), and bone marrows (BM) of B6, IL-2R{alpha} KO, and DM micea

 
Interestingly, there is a significant increase in CD4+ T cells in the bone marrow (from 1.3 x 106 in femurs in B6 mice to 3 x 106 in IL-2R{alpha} KO mice and 4 x 106 in DM mice). In contrast to normal B6 control, a high proportion of the CD4+ T cells in the bone marrow of IL-2R{alpha} KO and DM mice expressed CD69 (data not shown). This may explain why a 2- to 3-fold increase in CD4+ T cells is sufficient to induce disease phenotype in IL-2R{alpha} KO and DM mice. Alternatively, the bone marrow is highly sensitive to inflammation-mediated effects of activated T cells. The reason for the accumulation of CD4+ T cells is unclear at present, but it is consistent with the marked abnormalities observed in the bone marrow and the importance of CD4+ T cells to induce the disease phenotypes as demonstrated by anti-CD4 mAb blocking and adoptive transfer experiments (14).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The consequence of the immune defect in IL-2R{alpha} targeted mutation is very complex. Recent advance in Treg cell research strongly suggests that many of the various immune phenotypes observed in IL-2R{alpha} KO mice are initiated by the lifelong absence of the CD4+CD25+ Treg cells. In the absence of this population of Treg cells, lymphocytes in young mice are polyclonally activated and certain tissues are infiltrated by these activated lymphocytes, leading to tissue inflammation and pathology. We characterized the inflammatory responses and the Fas-based tissue damage in various tissues/organs. Among them, the lung and colon pathology are severe and may contribute to mortality. Importantly, we also found that bone marrow is a major target of infiltration by activated T cells and certain immune phenotypes observed in the IL-2R{alpha} KO mice is likely the consequence of this process. We demonstrated that along with bone marrow infiltration of activated T cells, IL-2R{alpha} KO mice developed anemia and blocked the formation of pre-B/immature B cells, the latter event was followed by B cell depletion in peripheral lymphoid tissues and the reduction of serum IgG level in aged mice. Our study covers the lifespan of the mice starting from genotyping at 4 wk of age to the point where the mice are moribund. A schematic presentation of these pathways/processes is shown in Fig. 10, which describes our view on the immune system biology in IL-2R{alpha} KO mice based on the roles of Treg cells and Fas in this system (see figure legend). This hypothesis is supported by the fact that these phenotypes are progressive and age-dependent and that similar phenotypes were observed in IL-2 KO mice and IL-2R{beta} KO mice that also lack the CD4+CD25+ Treg cells (8, 10, 12, 14). Our data also demonstrated the dominant feature of Treg cell abnormality over Fas mutation because the majority of the phenotypes in the DM mice resemble IL-2R{alpha} KO mice (multiorgan inflammation and early increase in serum IgG, etc.) but not the B6.lpr mice (accumulation of the abnormal double-negative T cells (data not shown) and late increase in serum IgG level).



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  		FIGURE 10. Roles of Treg cells and Fas played in the immune system biology in IL-2R{alpha} KO mice: a schematic presentation. In newborn IL-2R{alpha} KO mouse, thymocyte differentiation produces CD4+CD25– T cells but not CD4+CD25+ Treg cells. In the periphery, CD4+CD25– T cells are spontaneously and polyclonally activated and expanded in the absence of Treg cells. Some of the autoimmune T cells go to various tissues/organs and cause inflammation and Fas-based tissue damages. Some autoimmune T cells go to bone marrow where they cause bone marrow depression, leading to inhibition of erythropoiesis and B cell lymphopoiesis that contribute, respectively, to anemia and B cell deletion. We have not yet determined whether the generation of thymocyte precursors is inhibited.

 
B cell deletion has been reported in a few old IL-2 KO mice (9, 12) and IL-2R{beta} KO mice (14) but not IL-2R{alpha} KO mice (13). B cell deletion in IL-2 KO mice requires T cells because it was not observed in nude IL-2 KO mice, and B cells in nude mice were deleted upon adoptive transfer of T cells from IL-2 KO mice (12). B cell deletion in IL-2R{beta} KO mice requires CD4+ T cells because it was prevented by anti-CD4 mAb treatment (14). In both IL-2 KO mice and IL-2R{beta} KO mice, the differentiation step from pro-B cells to pre-B/immature B cells in the bone marrow was blocked (12, 14). This blockade was demonstrated in IL-2R{alpha} KO mice in the present study. However, B cell development was apparently normal in mice before 4–8 wk of age, arguing against the interpretation that IL-2/IL-2R signaling is essential for B cell development (12). Collectively, these studies suggest that the autoimmune expansion of CD4+ T cells must have occurred and they are responsible for the B cell deletion. It was hypothesized that these activated T cells infiltrate bone marrow and interfere with the B cell differentiation process (12). We showed that in IL-2R{alpha} KO mice, the absolute number of CD4+ T cells infiltrated in bone marrow was significantly increased, providing strong supporting evidence that infiltration of lymphocytes into the bone marrow is affecting the generation of pre-B/immature B cells. A number of mechanisms have been suggested, including T cell-mediated killing of B cells (12, 24). Our study demonstrates that the bone marrow B cell deletion phenotype in IL-2R{alpha} KO mice is Fas-independent because the differentiation from pro-B cells to pre-B/immature B cells is also observed in the DM mice. Moreover, the absolute number of B cells in the peripheral lymphoid tissues was reduced significantly both in old IL-2R{alpha} KO and DM mice. These observations indicate that B cell depletion in the periphery is mostly Fas-independent. They also suggest that the B cell differentiation block in the bone marrow is the main mechanism responsible for the peripheral B cell depletion in older mice.

We also observed a remarkable increase in CD8+ T cells in the IL-2R{alpha} KO and DM mice. This phenotype is not observed in IL-2 KO mice (data not shown) and not reported in IL-2R{beta} KO mice (14). This phenotype is not described in detail in this study because it is intrinsic to IL-2R{alpha} KO and independent from Treg expression defect. In IL-2R{alpha} KO mice, the IL-2R/IL-15R {beta}-chain expression is up-regulated (25) and the {beta}{gamma} chains are more available for pairing with IL-15R{alpha} to form the high affinity IL-15R. The overexpression of high affinity IL-15R and the omnipresence of IL-15 in virtually all tissues (26, 27) could provide a favorable condition for T cell expansion. Additionally, IL-15/IL-15R interaction appears to affect preferentially CD8+ T cell homeostasis by providing a strong antiapoptotic signal that favors CD8+ T cell survival (28). In support of this, there is a 40–50% reduction in the CD8+ T cell number in the lymph nodes and spleens of IL-15 KO and IL-15R KO mice compared with their normal counterparts (29, 30). Our observation of a remarkable increase in CD8+ T cells in IL-2R{alpha} KO mice provides a reciprocal situation to IL-15 KO mice and IL-15R{alpha} KO mice.

Increase in CD4+ T cell numbers was not observed in the lymphoid tissues of IL-2R{alpha} KO mice except in bone marrow. It is likely that many activated T cells have already infiltrated into various inflamed tissues. Therefore, the apparent lack of an increase in CD4+ T cells cannot be taken as evidence of lacking T cell activation and expansion. In support of this interpretation, the proportion of activated phenotype, as defined by expression of activation marker CD69, increased 2.5- to 4-fold in the lymphoid tissues of IL-2R{alpha} KO and DM mice in comparison with B6 counterparts (Fig. 3). This is observed in both CD4+ and CD8+ T cell populations. It is important to note that in the bone marrow of IL-2R{alpha} KO and DM mice, both the total number of CD4+ T cells and the percentage of activated phenotype of CD4+ T cells were increased in comparison to B6 control, consistent with the interpretation that these infiltrated T cells are responsible for the suppression of bone marrow function observed in these mice.

The inflamed tissue studied most in IL-2 KO, IL-2R{alpha} KO, and IL-2R{beta} KO mice is colon, and ulcerative colitis has been considered as a major cause and predictor for mortality. We have previously shown that lpr mutation does not inhibit colon inflammation but inhibits colon epithelial cell apoptosis and colon damage in IL-2 KO mice (21). We attributed this protection of colon damage to the prevention of mortality. The data presented in the present study indicate inflammation in multiple tissues so that attribution of colitis to mortality may be simplistic at least in the case of IL-2R{alpha} KO mice. Particularly, the damage in the lung is so severe that it is likely to contribute to the mortality observed in IL-2R{alpha} KO mice. A severe inflammation in the lung as well as several phenotypes described herein were also reported for a patient bearing a deletion mutant IL-2R{alpha} gene (31). Inflammation in lung, pancreas, and colon was also reported in gnotobiotic IL-2 KO mice (10). Despite the protection of tissue cell apoptosis by Fas mutation, mortality was only protected partially. Thus, mortality in IL-2R{alpha} KO mice involves both Fas-dependent and Fas-independent mechanisms.

In summary, we have characterized in great detail a number of phenotypes in IL-2R{alpha} KO mice and determined the role of Fas played in these phenotypes. In addition to colon, we have identified new tissues that are inflamed and some that are damaged by Fas-based mechanisms. Among them, we identified bone marrow as a target of autoimmune T cells that are expanded in the absence of the CD4+CD25+ Treg cells. Data presented suggest that B cell deletion and anemia are the consequences of the infiltration of these autoimmune T cells that suppress bone marrow function. Of great significance is that we described many of these phenotypes in a time- and age-dependent fashion, thus, providing a lifelong and global picture of the immune system biology of IL-2R{alpha} KO mice. In this view, the effect of Treg cells is largely independent from Fas mutation because FasL induction and Fas up-regulation occurs subsequent to the polyclonal T cell activation resulted from lifelong absence of the CD4+CD25+ Treg cells. Accordingly, the role of Fas on the immune system biology of IL-2R{alpha} KO mice becomes evident only in older mice when the autoimmune inflammation is fully developed. Indeed, Fas mutation did not block inflammation of various organs and only affected phenotypes of inflammation that are Fas-dependent such as apoptosis and chemotaxis. The immune system biology in IL-2R{alpha} KO mice, as depicted in Fig. 10, is based on the loss of the CD4+CD25+ Treg cells and through information gathered from both in vitro and in vivo research. This scheme provides a general map for future studies and for the refinement of the immune system biology in mice lacking CD4+CD25+ Treg cells.


   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 Address correspondence and reprint requests to Dr. Shyr-Te Ju, Division of Rheumatology and Immunology, Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908-0412. E-mail address: sj8r{at}virginia.edu Back

2 Abbreviations used in this paper: Treg, regulatory T; KO, knockout. Back

Received for publication February 28, 2005. Accepted for publication May 5, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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