Originally published online as doi:10.1189/jlb.1006638 on April 24, 2007
Published online before print April 24, 2007
(Journal of Leukocyte Biology. 2007;82:429-435.)
© 2007
by Society for Leukocyte Biology
Large defects of type I allergic response in telomerase reverse transcriptase knockout mice
Azusa Ujike-Asai*,
Aki Okada*,
Yuchen Du*,
Mitsuo Maruyama*,
Xunmei Yuan
,
Fuyuki Ishikawa,
Yoshiharu Motoo
,
Kenichi Isobe*,
and
Hideo Nakajima*,,1
* Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Japan;
Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kyoto, Japan;
Department of Medical Oncology, Kanazawa Medical University, Uchinada, Japan; and
Department of Immunology, Graduate School of Medicine, Nagoya University, Nagoya, Japan
1 Correspondence: Department of Medical Oncology, Kanazawa Medical University, Daigaku 1-1, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. E-mail: hideonak{at}kanazawa-med.ac.jp
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ABSTRACT
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Telomerase is critically important for the maintenance of a constant telomere length, which in turn, is related to the concepts of longevity and oncogenesis. In addition, it has been well documented that telomerase activity is expressed in immune cells in a highly regulated manner. We have studied systemic anaphylaxis in mouse telomerase reverse transcriptase knockout (mTERT–/–) mice to understand the significance of telomerase activity and telomere stability in mast cells, which induce a type I allergic response. Compared with wild-type mice, mTERT–/– mice displayed largely attenuated, IgE-mediated, passive anaphylactic responses, which were observed even in the early generations of mTERT–/– mice, and had decreased numbers of mast cells in vivo and impaired development of bone marrow-derived mast cells (BMMCs) induced by IL-3 or stem cell factor in vitro. Moreover, in mTERT–/– mice, BMMCs exhibited a large morphology and low proliferation rate, while they possessed a comparable degranulation capacity and cell surface expression level of c-kit and Fc
RI. These findings imply that telomerase activity has a definitive impact on the type I allergic response by altering the character of effecter mast cells.
Key Words: anaphylaxis • mast cell • TERT
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INTRODUCTION
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Telomerase is a ribonucleoprotein enzyme, which adds telomeric repeats to chromosome ends to extend or maintain telomere length, which consequently avoids replicative senescence [1
]. Telomerase consists of two essential subunits, the template RNA [telomerase RNA (TR)] and the catalytic subunit [telomerase reverse transcriptase (TERT)]. A defect of TR or TERT results in a lack of telomerase activity.
In knockout (KO) mice with TR deletion mutants (mTR–/–) on a C57BL/6 background, sterility, splenic atrophy, reduced proliferative capacity of B and T cells, abnormal hematological parameters, and progressive loss of organismal viability were observed coincident with telomere shortening in the late generations {Generations 4–6 (G4–G6) [2
]}. In addition, the survival rate of the late generations decreased dramatically with age in comparison with wild-type (WT) mice. Splenocytes in the late generations of mTR–/– mice displayed telomere shortening, genomic instability, and a decreased number of germinal centers after immunization [3
]. Recently, a number of studies have investigated the telomere length and telomerase expression in leukocytes during the activation, differentiation, and maturation process in combination with aging [4
, 5
]. As the function of the immune system is highly dependent on the capacity for extensive cell division and expansion of immune cells, these studies may shed new light on the regulation of the immune system. Although deep involvement of the telomere structure and telomerase activity in immune reactions is easily assumed, their precise role and physiological function remain unclear.
Previously, mice with a TERT deletion (mTERT–/–) have been reported as a similar phenotype to mTR–/– mice, which showed apparently normal macroscopic or microscopic findings in the early generations but developed multiple abnormalities in the late generations [6
]. Here, we report that mTERT–/– mice display large anaphylactic response defects even in the early generations, thereby indicating a pivotal role for this enzyme in allergic reactions.
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MATERIALS AND METHODS
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Antibodies
Rat anti-mouse Fc
RIIB/III (2.4G2) and mouse anti-TNP IgE (IGELa2) were purified from the hybridoma soup by ion exchange chromatography on DEAE cellulose (Merck, Whitehouse Station, NJ, USA) and by affinity isolation with a protein G column.
Animals
C57BL/6 mice were purchased from Charles River Japan (Yokohama), and 6- to 24-week-old mice were used as WT mice. Male and female mTERT+/– mice with a C57BL/6 genetic background were described previously [6
]. G1 TERT–/– mice were obtained by crossing mTERT+/– and mTERT+/– mice. G1 TERT–/– mice were mated to generate G2 mice, which were bred further to obtain successive generations. Mice were housed in the Animal Unit of the National Institute for Longevity Sciences (Aichi, Japan), an environmentally controlled and specific, pathogen-free facility, and experiments were performed according to guidelines for experimental animals defined by the facility.
Induction of passive systemic anaphylaxis (PSA) and monitoring of rectal temperature
Mouse IgE anti-TNP mAb (1 mg/kg) were administered i.v. through the tail vein, and after 24 h, mice were injected with 50 mg/kg TNP4-OVA. Changes in body temperature associated with systemic anaphylaxis were monitored by measuring changes in rectal temperature using a rectal probe coupled to a digital thermometer (Natsume Seisakusyo Co., Tokyo, Japan) as described elsewhere [7
8
9
].
Serum histamine level
Mouse serum was obtained from the subocular plexus 5 min after antigen challenge. Histamine levels in the plasma samples from 10 mice were quantified using ELISA and were performed by SRL, Inc. (Tokyo, Japan).
Bone marrow-derived mast cell (BMMC) culture
c-kit-positive cells in BM cells were positively selected by MACS sorting using c-kit microbeads (Miltenyi Biotec, Gladbach, Germany). To keep mast cells in culture, BM c-kit+ cells were cultured at 3–5 x 105 cells/ml in a medium containing 66 ng/ml recombinant mouse stem cell factor (rmSCF; Peprotech, Rocky Hill, NJ, USA) and/or 5 ng/ml rmIL-3 (R&D Systems Ltd., Abingdon, UK) with 10% FCS (Sigma, Poole, UK). Medium was changed every 3 or 4 days. When more than 90% of cells were differentiated into mast cells 
3 weeks later, they were used for the experiments.
Measurement of telomerase activity
Telomerase activity was measured with the telomerase PCR-ELISA kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions as described previously [10
]. In brief, the cell extract (representing 103 cells) was incubated for 30 min at 25°C in a reaction mixture of telomerase and then subjected to 40 cycles of PCR amplification (94°C for 30 s, 58°C for 30 s, and 72°C for 90 s). PCR products were detected by ELISA, and the absorbance of samples was measured at 450 nm with a Microtiter plate reader (Bio-Rad, Hercules, CA, USA). All assays were performed in triplicate. The level of telomerase activity in the positive cell extract supplied in the kit was set to 100%, and the relative specific telomerase activity (RTA) of each extract was expressed as a percentage of the positive control standard.
Mast cell degranulation
BMMCs were adjusted to 2 x 105 cells/ml in culture medium and labeled with 1 µCi/ml [3H]-serotonin (Amersham Biosciences, Uppsala, Sweden) overnight at 37°C. Then, cells were washed three times and resuspended at 2 x 105 cells/ml in the medium. Degranulation was carried out in 96-well plates after incubation with 5 µg/ml anti-TNP IgE, followed by cross-linking with 10 µg/ml TNP-OVA for 1 h at 37°C. Cell-free supernatants were collected and counted in a scintillation counter (experimental release). Total counts per 50 µl-labeled cells (total release) and spontaneously released counts (spontaneous release) were also determined to calculate the percent of serotonin release as follows: [(experimental release–spontaneous release)/(total release–spontaneous release)x100].
Flow cytometry
For flow cytometric analysis, we used the following mAb: FITC-, PE-, or biotin-conjugated, anti-mouse IgE (R35-72), anti-c-kit (2B8), and anti-TNP IgE. All antibodies were purchased from BD PharMingen (San Diego, CA, USA). Cell surface staining was carried out using standard techniques. Two million cells were incubated for 30 min at 4°C with antibodies, and flow cytometric analysis was performed with FACSCalibur using CellQuest software (BD PharMingen). Dead cells were eliminated from the analysis on the basis of propidium iodide (PI) staining.
Apoptosis assay
BMMCs were deprived of IL-3 for 24 h and cultured in the presence or absence of IL-3 for an additional 48 h. Also, 0.01 mM dexamethasone (DXM) was used to induce apoptosis. After incubation, cells were stained with PI and Annexin V using Annexin V-FITC apoptosis detection kit I (BD PharMingen) and subjected to FACS analysis.
Proliferative response of mast cells
BMMCs were activated with rmIL-3, rmSCF, or rmIL-3 plus rmSCF. After 24 h of stimulation, proliferation was determined by [3H]-thymidine uptake as described previously [11
]. In brief, culture wells were pulsed with [3H]-thymidine (1 µCi) for 12 h before being collected on glass fiber filters and counted in a scintillation counter. Experiments were performed in quadruplicate.
Senescence-associated β-galactosidase activity (SA-β-Gal)
To investigate expression of SA-β-Gal in BMMCs, the cultured cells were stained histochemically with β-Gal (pH 6.0) using the Senescence β-Galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA). After 2 h incubation, samples were developed and observed under the microscope.
Histological study
Cultured cells (2x105) were placed on slides and fixed in 4% paraformaldehyde. Tissues from mouse ear were fixed in 10% (vol/vol) neutral-buffered formalin and embedded in paraffin. The specimens were sectioned at 5 µm. Then, prepared slides were stained with 0.5% toluidine blue (pH 4.0). Mast cells were identified by the presence of abundant numbers of metachromatically stained granules. The number of mast cells was counted under a light microscope (/mm2).
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RESULTS
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Attenuated IgE-mediated systemic anaphylaxis in mTERT–/– mice
We investigated IgE-mediated PSA using the early generation (G1, G2, G3) of mTERT–/– mice. To elicit the anaphylactic response, mice were sensitized with IgE specific for TNP, followed by i.v. administration of TNP-OVA. In all generations tested, mTERT–/– mice showed a largely attenuated anaphylactic response as compared with WT mice, and with successive generations, the attenuation became greater (Fig. 1
). Thus, a marked diminution of the IgE-dependent PSA reaction was observed, even in the early generations of mTERT–/– mice. We also examined serum histamine levels during anaphylaxis by ELISA, but no significant difference was observed between WT and mTERT–/– mice (WT: 13,267±3786 nM; mTERT–/–: 15,333±3164 nM, mean±SD; P=0.51).
High levels of telomerase activity in mouse mast cell
Mast cell is the effecter cell of systemic anaphylaxis through the binding of IgE to its surface receptor FcRI. First, we measured the telomerase activity of mast cells during differentiation from WT BM cells maintained under standard culture conditions. BMMC, determined by c-kit+ cells, showed a rapid increase of telomerase activity immediately after starting culture in the presence of IL-3 (Fig. 2A
). They reached a maximum after 3 days of culture and sustained high levels of activity thereafter. It has been reported that differentiation and maturation of mast cells are influenced by various microenvironments including numerous growth factors such as IL-3, IL-4, IL-9, IL-10, nerve growth factor, and the fibroblast-derived SCF [12
]. In addition, monomeric IgE leads to a potent production of cytokines from BMMCs and enhances BMMC survival by preventing the apoptosis of BMMCs [13
]. To investigate the effects of these cytokines and monomeric IgE on telomerase activity, BMMCs were restimulated with IL-3, SCF, or monomeric IgE in various combinations. However, no significant alteration of telomerase activity was observed in BMMC with these stimulations (Fig. 2B)
.
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Figure 2. Telomerase activities of BMMC from WT mice. (A) Time course of telomerase activity in IL-3-induced mast cells from WT BM cells. Telomerase activity was measured by telomerase PCR ELISA. RTA was calculated as a percentage of the positive control standard. (B) Telomerase activity of BMMC after stimulation. Established BMMCs from WT mice were stimulated in the presence of IL-3 (5 ng/ml), SCF (50 ng/ml), or anti-TNP IgE (5 µg/ml) plus TNP-OVA (1 µg/ml). After 24 h, telomerase activities were measured.
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Characteristics of mast cells in mTERT–/– mice
Mast cells degranulate and release chemical mediators when FcRI on the cell surface is cross-linked by IgE and antigens. Furthermore, monomeric IgE stimulation enhances cell surface expression of FcRI on mast cells. To clarify whether a loss of telomerase activity affects these functions, we first measured granular release from mast cells. As shown in Figure 3A
, after 10 min incubation with IgE followed by 1 h stimulation with antigen, the degree of serotonin release from BMMCs of mTERT–/– G2 mice was the same as that of WT mice. After a longer incubation period of 24 h, the serotonin release from BMMCs increased in WT and mTERT–/– mice but exhibited no significant difference between them. These data indicated that the disruption of the TERT gene did not alter the mast cell function of FcRI-mediated degranulation.
Next, we performed flow cytometric analysis to examine whether deletion of the TERT gene influences FcRI and c-kit expression on mast cells. As shown in Figure 3B
, the expression levels of FcRI and c-kit on BMMCs from mTERT–/– G2 mice were comparable with those from WT mice. Similarly, peritoneal mast cells isolated from mTERT–/– G2 mice and WT mice, 24 h after i.v. administration of IgE, displayed equivalent levels of FcRI and c-kit expression on the cell surface. Altogether, flow cytometric analysis revealed no considerable difference in the cell surface markers on mast cells between mTERT–/– and WT mice.
Impaired proliferation and development of mast cells in mTERT–/– mice in vitro and in vivo
IL-3 and SCF are essential factors in mast cell differentiation in vitro as well as in vivo. First, we examined the induction of BMMCs from mTERT–/– G2 mice and WT mice in the presence of IL-3 or SCF using a standard technique. Judging from the number of mast cells expanded, BM cells from mTERT–/– G2 mice showed a limited capacity for development into BMMCs (Fig. 4A
). Especially, mTERT–/– G2 BM cells died in 3 weeks under culture with SCF. As impaired development of BMMCs was observed in mTERT–/– mice, we investigated the apoptotic tendency of BMMCs. Apoptosis was induced by cytokine (IL-3) depletion or DXM stimulation, the standard technique to trigger cell death of leukocytes. In all conditions tested, BMMCs from mTERT–/– mice exhibited slightly higher rates of cell death in the early phase of apoptosis (Annexin-V+/PI–) and the late phase of apoptosis or necrosis (Annexin-V+/PI+), although the differences were not significant (Fig. 4B)
. Next, we examined the proliferative responses of BMMCs. In Figure 4C
, BMMCs from mTERT–/– G2 mice displayed a largely reduced proliferation rate as compared with those from WT mice in response to IL-3, SCF, or both.
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Figure 4. Impaired development and proliferation of mast cells in mTERT–/– mice. (A) SCF- and IL-3-induced development of mast cells from BM cells. WT or mTERT–/– BM cells were cultured in the presence of IL-3 or SCF to induce mast cells during the indicated number of days. The number of live cells was determined with trypan blue dye exclusion. (B) Apoptotic tendency of BMMCs. After 24 h cytokine depletion, BMMCs were cultured with (IL3) or without (None) cytokine for an additional 48 h. Also, 0.01 mM DXM was added to induce apoptosis. Then, BMMCs were stained with Annexin-V (horizontal axis) and PI (vertical axis). Annexin-V+/PI– cells: early phase of apoptosis; Annexin-V+/PI+ cells: late phase of apoptosis or necrosis. A representative of five independent experiments is shown. (C) Proliferative response of BMMCs in cytokine stimulation. IL-3-induced BMMCs from WT (open bars) and mTERT–/– mice (solid bars) were cultured in the presence of the indicated concentration of IL-3, SCF, or both. The data (mean±SD in triplicate), which were representative of three independent experiments, are shown.
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Morphological features of mast cells
By microscopic analysis, the development of mast cells was judged by toluidine blue staining. The presence of acid toluidine blue-positive, granule-containing cells in the culture of BM-derived cells was observed in WT and mTERT–/– mice. However, most BMMCs from mTERT–/– mice displayed an enlarged morphology with big nucleus and vacuoles, as often seen in senescent cells (Fig. 5A
, left). These large cells were also stained deeply with β-Gal under a pH 6.0 condition, which indicates SA-β-Gal activity (Fig. 5A
, right). To investigate the development of mast cells in vivo, we performed a histopathological analysis. Each ear of WT and mTERT–/– mice was fixed and stained with acid toluidine blue. As shown in Figure 5
, B and C, the number of mast cells in ear dermis was significantly lower in mTERT–/– mice. These results indicated that the lack of mTERT expression largely inhibits the proliferation and development of mast cells in vitro and also in vivo.
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Figure 5. Morphological features of mast cells in mTERT–/– mice. (A) WT or mTERT–/– BMMCs were examined histologically after staining with toluidine blue (original, x400, left) and SA-β-Gal (original, x600, right). (B) Comparison of dermal mast cells in the ear between WT and mTERT–/– mice. Mast cells were detected with toluidine blue staining as the metachromatic cells indicated with red arrows (upper, original, x300; lower, original, x400). (C) Mast cell number in ear dermis before and after PSA. Densities of dermal mast cells were calculated by counting cells under light microscopy in 15 different sections each from four different mice. The results are expressed as mean ± SE (P<0.02 and P=0.02).
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DISCUSSION
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Leukocytes derive from BM hematopoietic stem cells and circulate in blood and tissues. Their distinctive process of differentiation and rapid proliferation in immune responses require involvement of numerous biological factors, including telomerase activity and telomere maintenance, which are implicated in the regulation of the replicative lifespan of dividing cells. In addition, in aging, the attrition of telomere length in leukocytes and decline of immune functions are observed [2
, 3
]. Thus, the function of the immune system seems deeply associated with telomere length and telomerase activity in leukocytes during differentiation, activation, and also aging. Accumulating data derived from analysis of telomere length and telomerase expression in hematopoietic stem cells and lymphoid lineage cells indicate that telomerase activity is highly regulated in hematopoietic stem cells and lymphoid cells and plays a pivotal role in the telomere maintenance and replicative lifespan of cells [4
, 5
], although its precise physiological function remains to be elucidated. Contrary to the considerable information available on lymphoid lineage cells such as T and B cells, few reports have addressed the issue of telomere length and telomerase activity in myeloid lineage cells, which are involved mainly in innate immunoresponses and tend to have a short lifespan and high turnover rate [10
, 14
].
In this report, we analyze the impact of telomerase activity on anaphylactic responses, which are mediated by mast cells. It has been reported that telomerase is expressed during human mast cell development from hematopoietic stem cells and mast cell progenitor cells. Although mast cell progenitors showed a rapid increase in telomerase activity preceding proliferation in response to SCF and IL-3 or IL-6, the induction was transient, and telomerase activity declined to basal levels. No telomerase activity was detected in human mature mast cells [14
]. To clarify the physiological role of telomerase expression in mast cells, we examined anaphylactic responses and mast cell functions using TERT KO mice. We chose to evaluate a passive rather than an active model of anaphylactic response, as mTERT–/– mice might exhibit dampened, humoral immune responses as a result of impairment of IgE production from B cells. To elicit an anaphylactic response, mice were injected with IgE specific for TNP, followed by i.v. administration of TNP-OVA. In this study, significantly attenuated IgE-mediated PSA was observed even in the first generation (G1) of mTERT–/– mice (Fig. 1)
. The defects in the anaphylactic response became more apparent with successive generations in the mTERT–/– mice. As a mouse has a long telomere length, mTR–/– and mTERT–/– mice in the early generations have been reported to show no gross abnormality caused by telomere shortening [2
, 15
]. In mTR–/– mice, reduced proliferative capacity of B and T cells, poor antibody responses, and a decreased number of germinal centers and splenic atrophy were observed only in the late generations (G4–G6) [2
, 3
]. These findings suggest that mast cell-mediated anaphylactic responses are sensitive to the loss of telomerase expression.
Differing from human mast cells, BMMCs from WT mice showed persistently high telomerase activity from an early stage of induction, and activity was not affected by stimulation with IL-3, SCF, or IgE (Fig. 2)
. The fact that mast cells maintained high telomerase activity during development from BM cells prompted us to examine whether deletion of mTERT would influence the characteristics of mast cells such as granular formation, degranulation capacity, and cell surface expression. However, we could not demonstrate any significant differences in degranulation capacity or cell surface expression levels of c-kit and FcRI between WT and mTERT–/– mast cells (Fig. 3)
. Conversely, in the absence of telomerase expression, the development of mast cells from BM cells and the proliferation in response to IL-3 and SCF were impaired severely (Fig. 4)
. In microscopic analysis, WT and mTERT–/– BMMCs possessed abundant acid toluidine blue-positive granules equally, but mTERT–/– BMMCs displayed morphological features of senescence such as enlargement of cells and expression of SA-β-Gal activity. Histologically, mTERT–/– mice carried a smaller number of mast cells in ear dermis, reflecting their lower proliferation, higher turnover rate, or possibly, reduced migration of mast cells (Fig. 5)
. Taken together, these results suggest that a defect of telomerase expression interferes with the proper development and proliferation of mast cells, resulting in a reduced number of mast cells and largely attenuated anaphylactic responses in vivo.
The treatment of allergic disease, caused by foods, drugs, injections, insect stings, or inhalations, has assumed increasing importance. The most dangerous allergic reaction is anaphylaxis, which can be a life-threatening emergency. A number of drugs such as antihistamines, adrenalin, and steroids have been developed to manage anaphylaxis, but it still is not easy to predict, prevent, and treat. In the present study, we demonstrate the importance of telomerase activity in anaphylaxis, which may help to understand the age-related alteration of allergic responses and also suggest new, therapeutic strategies to control allergic responses.
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ACKNOWLEDGEMENTS
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This study was supported by the Uehara Memorial Foundation, Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists, Hokkoku Gan-Kikin, a Research Grant for Longevity Sciences (13C-1, 16A-2) from the Ministry of Health and Welfare, the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, and a grant from the Japan Health Science Foundation. A. U-A. is a research fellow of the Japan Society for the Promotion of Science.
Received October 18, 2007;
revised March 28, 2007;
accepted March 29, 2007.
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ABSTRACT
INTRODUCTION
) or early generations of mTERT–/– mice (
; G1:A, G2:B, G3:C). After mice received 1 mg/kg anti-TNP IgE i.v., 50 mg/kg TNP-OVA was injected 24 h later to induce systemic anaphylaxis. Data are shown as mean ± SD. *, P < 0.05.