- Email: [email protected]
Diminished cytokines gene expression in lymphoid organs of healthy aged rats
Cytokine 54 (2011) 24–28
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Diminished cytokines gene expression in lymphoid organs of healthy aged rats _ Maria Pachówka a,⇑, Jolanta Makula b, Grazyna Korczak-Kowalska a,b a b
Department of Immunology, University of Warsaw, Warsaw, Poland Transplantation Institute, Medical University of Warsaw, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 1 September 2010 Accepted 9 November 2010 Available online 6 January 2011 Keywords: Aging Cytokine Gene expression Lymphoid tissues Real-time PCR
a b s t r a c t Immunuosenescence-related dysregulation of cytokine production is not fully understood and the roles of cytokines in organ aging have been insufficiently studied. This work aimed to compare the expression of interleukin-2 (IL-2), IL-4, IL-6, interferon-gamma (IFNc), and transforming growth factor-beta (TGFb) in lymphoid organs of young (3 months; n = 10), adult (1 year; n = 10), and aged (2 years; n = 7) rats under healthy, steady-state conditions. Cytokine mRNA levels were determined with TaqMan real-time PCR. In the spleen, all cytokine expression gradually declined with age (for representative cytokine IL-2 averages dCt in spleens of 3 months, 1 year and 2 years rats were 13,645, 13,19 and 15,470, respectively). In lymph nodes, all cytokines except TGF-b showed markedly upregulated expression in adult compared to young rats, but all were expressed at very low levels in aged rats. In bone marrow, adult animals showed enhanced IL-2, IFNc, and TGFb expression, but similar IL-4 and IL-6 expression, compared to young rats. Bone marrow of aged rats showed very low expression of all the measured cytokines. Our results demonstrated that changes occurred unevenly with aging in different immune system compartments. Ó 2011 Published by Elsevier Ltd.
1. Introduction The function of the immune system declines with age. Thus, it follows that the complex cytokine network that orchestrates cell–cell interactions is also affected by age. Age-related changes in cytokine production have been investigated previously in both humans and rodents [1–3]. However, most previous studies were performed with either peripheral blood cells isolated from humans or with lymphoid organ cells isolated from rodents (e.g., spleen or lymph nodes). These isolates were typically cultured in vitro, stimulated with mitogens (antigens or polyclonal activators), and measured based on secreted cytokines in cell supernatants. Due to different culture conditions, stimulation methods, and measurement techniques, variable results have been reported. Therefore, in order to determine the in vivo changes in cytokine production that take place during the aging process, in this study, we used lymphoid tissue homogenates from healthy young, adult, and aged rats. The aim of this study was to study mRNA expression of cytokines in lymphoid organs (lymph nodes, spleen, and bone marrow) under healthy conditions. We studied rats with no vaccinations or immunizations in order to focus on the natural processes that take place during aging. Moreover, we did not isolate lymphocytes, but ⇑ Corresponding author. Address: Miecznikowa 1, 02-096 Warsaw, Poland. Tel.: +48 22 55 41 127; fax: +48 22 55 41 203. E-mail address: [email protected] (M. Pachówka). 1043-4666/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.cyto.2010.11.007
instead measured changes in cytokine expression in whole tissue homogenates. This may provide clues to in vivo production of cytokines in the context of the local tissue environment. We used TaqMan real-time PCR to analyze the expression of IL2, IFNc, IL-4, IL-6, and TGFb in lymph nodes, spleens, and bone marrow under steady-state conditions in young, adult, and aged rats. We found that each tissue exhibited different patterns of aging. In the period from 3 months to adulthood (1 year old), the lymph node cytokine production, except for TGFb, increased markedly; the spleen cytokine production declined gradually throughout life; and in bone marrow, the IL-2, IFNc, and TGFb production slightly increased with age, but IL-4 and IL-6 production remained stable. In aged rats (2 years old), all tissues exhibited very low numbers of transcripts for all tested cytokines. 2. Materials and methods 2.1. Animals Female Lewis rats were bred in the animal facilities of the Department of Experimental and Clinical Medicine at the Polish Academy of Sciences (Warsaw, Poland). Animals were divided into three groups based on their age. Three-months-old female rats represented the young group (n = 10), 1-year-old female rats represented the adult group (n = 10), and 2-years-old female rats represented the old group (n = 7). The animals were maintained at a stable temperature (23 ± 2 °C) and were provided with an
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unlimited and uninterrupted supply of food and water. Axillary lymph nodes and spleens were isolated and preserved in 70 °C. Bone marrow was isolated from thigh bone and RNA isolation was performed immediately after. All procedures involving animals were reviewed and approved by the First Warsaw Local Ethical Commission for Animal Experimentation (No. 642/2006). All experimental manipulations described herein were performed in strict accordance with the recommendation determined by the ethical commission. 2.2. RNA isolation Lymph nodes, spleens and bone marrows were homogenized in RLT lysis buffer from the RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA was isolated according to the manufacturer’s instructions. 2.3. Reverse transcription Complementary strand DNA was synthesized by combining 2 lg of RNA with 1 ll of 10 mM dNTP and 1 ll of 50 ng/ll random hexamers (Invitrogen Life Technologies) in Eppendorf tubes (Axygen, Union City, USA). The suspension was brought up to a total volume of 10 ll with DEPC-treated water. Tubes were kept for 5 min in a Thermomixer Comfort heat block (Eppendorf, Hamburg, Germany). The tubes were then put on ice. Ten microliters of reaction mixture (Master Mix) was then added to each tube. Each aliquot of reaction mixture consisted of: 2 ll of 10 RT buffer, 2 ll of 0.1 mM DTT, 4 ll of 25 mM MgCl2, 1 ll of RNaseOUT (40 U/ll) and 1 ll of SuperScript III RT (200 U/ll) (Invitrogen Life Technologies). The tubes were kept at 25 °C for 10 min in the heating block. The temperature was then increased to 50 °C, and the tubes were kept in the heating block for another 50 min. For the last 5 min, the temperature was raised to 85 °C. 2.4. Real-time PCR The levels of IL-2, IFN-gamma, IL-4, IL-6, and TGF-beta transcripts were determined by real-time PCR. PCRs were performed in an ABI PRISM 7700 Sequence Detection System. Each of the 27 reaction mixtures contained: template cDNA, TaqMan Universal PCR Master Mix (Applied Biosystems, USA), and TaqMan gene expression assay for the genes of interest (Applied Biosystems, USA). Samples were normalized using rat GAPDH expression. The reaction was carried out under the following conditions: one cycle for 2 min at 50 °C and 10 min at 95 °C; and 40 cycles for 15 s at 95 °C and 1 min at 60 °C. Ct values representing the number of cycles at which the fluorescence value for each sample exceeded the threshold value were recorded. The expression level in each sample was compared with a calibrator. The following formula was applied: gene expression = 2DDCt In brief, relative quantification was performed using 2DDCt method. Reference gene for GAPDH was used. GAPDH as internal control was used for the normalization of the quantity of RNA. For normalization of the results Ct value for GAPDH was subtracted from Ct value for each of target genes (IL-2, IFNc, IL-4, IL-6, TGFb).
DCt ¼ Average CtTarget gene Average CtGAPDH The obtained difference (DCt) was then used to calculate DDCt with the formula:
DDCt ¼ DCt1-year-old=2-years-old rats DCt3-months-old rats The relative gene expression for cytokines normalized to an endogenous reference and relative to a calibrator was expressed as:
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Gene expression level ¼ 2DDCt Relative gene expression for targeted genes in control group (3-months-old rats) equals 1 (since 20 = 1). Relative gene expression for meassured cytokines in lymphoid tissues of 1-year-old and 2-years-old rats was presented as the fold change comparing to the control group [4,5]. 2.5. Statistical analysis All continuous data are expressed as mean ± standard deviation (SD). Analyses were performed to estimate differences using the Turkey test. A p value of <0.05 was considered to be statistically significant. For statistical analysis, only DCt values were used. For better interpretation, data were described in terms of relative gene expression. 3. Results 3.1. Relative gene expression of IL-2 in lymphoid tissues Aged (2-years old) rats exhibited the lowest IL-2 expression levels in lymph nodes, spleen, and bone marrow. In contrast, the relative gene expression of IL-2 was elevated in adult (1-year old) rats in all three examined organs. In particular, in adult rat lymph nodes, IL-2 gene expression was over 12-fold higher than that observed in young (3-months old) rat lymph nodes (Fig. 1). 3.2. Relative gene expression of IL-4 in lymphoid tissues The spleen exhibited a constant decline in IL-4 mRNA levels throughout the aging process. The spleens of aged rats exhibited the lowest relative IL-4 expression. The lymph nodes of adult rats exhibited very high IL-4 gene expression (over 23-fold higher) compared to those of young rats. The lymph nodes of aged rats exhibited significantly lower levels of IL-4 transcripts. The bone marrow of adult rats exhibited a slight increase in IL-4 gene expression compared to the bone marrow of young rats; but the IL-4 levels declined in the bone marrow of aged rats (Fig. 2). 3.3. Relative gene expression of IL-6 in lymphoid tissues Despite the fact that all the examined tissues from aged animals exhibited nearly undetectable IL-6 mRNA levels, the relative IL-6 expression in adult animals differed remarkably among different tissues. The lymph nodes of adult rats exhibited increased IL-6 mRNA levels compared to those of young rats. In contrast, the spleens of adult animals exhibited lower IL-6 mRNA levels compared to those of young rats. Finally, the bone marrow of adult rats exhibited IL-6 expression levels similar to that observed in young rats (Fig. 3). 3.4. Relative gene expression of IFNc in lymphoid tissues The relative gene expression of IFNc showed marked increases between 3 months and 1 year of age. In the lymph nodes and bone marrow, adult rats exhibited, respectively, over 11-fold and over 7-fold higher IFNc expression levels than those observed in young rats. In contrast, in the spleen, IFNc expression levels were similar in adult and young rats. However, with advancing age, significant reductions were observed in the relative expression of IFNc in all three tissues; the lowest level was observed in lymph lodes (Fig. 4).
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Fig. 1. Changes of IL-2 expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IL-2 gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IL-2 in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
Fig. 2. Changes of IL-4 expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IL-4 gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IL-4 in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
Fig. 3. Changes of IL-6 expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IL-6 gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IL-6 in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
3.5. Relative gene expression for TGFb in lymphoid tissues The relative TGFb expression pattern was similar to that observed for IFNc. In lymph nodes and bone marrow, adults rats exhibited in-
creased TGFb expression compared to that observed in young rats. In spleen, TGFb gene expression levels were very similar in young and adult rats. In addition, TGFb expression was significantly reduced in the spleen, lymph nodes, and bone marrow of aged rats (Fig. 5).
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Fig. 4. Changes of IFNc expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IFNc gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IFNc in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
Fig. 5. Changes of TGFb expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of TGFb gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for TGFb in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
4. Discussion In this study, we investigated the relative gene expression of five important cytokines, IL-2, IL-4, IL-6, IFNc, and TGFb, in the lymphoid organs of young, adult, and aged rats. We choose lymphoid organs, not peripheral blood mononuclear cells (PBMCs), because it is well known that lymphocytes circulating in blood are not representative for the whole immunological system. Hence, data based on PBMC investigations are not conclusive for the analyzing of the immune system activity. We found that aging affected cytokine mRNA expression differently in lymph nodes, spleen, and bone marrow. From youth, to adulthood, and from adulthood to later life, the spleen exhibited an age-related decline in cytokine production. In contrast, from youth to adulthood, the lymph nodes exhibited marked increases in the expression of all measured cytokines, except TGFb; moreover, in bone marrow, the TGFb, IL-2, and IFNc expression slightly rose and production of IL-4 and IL-6 remained stable. Then, from adulthood to later life, in lymph nodes, the expression of all cytokines dropped to nearly undetectable levels, and in bone marrow, we detected only very low levels of all cytokine mRNAs. Our finding that all aged tissues exhibited an age-related decline in cytokine production was inconsistent with the hypothesis that aging causes a decline in Th1 cytokine production (IL-2 and IFNc) and an increase in Th2 cytokine production (IL-4 and IL-6) [2]. Our results demonstrated that the predominant Th2 response
observed in the blood was not reflective of the organs. The discrepancy between our results and earlier findings might be explained by hypothesizing that aging does not diminish the ability of cells to produce cytokines, but aging diminishes the environmental stimulation. Thus, the mitogen stimulation provided in in vitro experiments is sufficient to induce isolated aged cells to secrete cytokines in cell culture. However, under normal aging conditions within the organ, there are no strong inducers that can stimulate cytokine secretion, either Th1 or Th2 cytokines. Our results suggested that, up to 1 year of age, the microenvironment promotes cytokine secretion in lymphoid organs, like lymph nodes. But, during the 2nd year of age, lymphoid tissues become devoid of cytokine transcripts; however, T cells and other cells retain the capability of cytokine production, as reported previously [3]. It is well known that lymphoid organs exhibit atrophy with aging. Morphological studies on human lymph nodes have revealed numerous age-associated changes, including increased adipose tissue content and a reduction in germinal center development [6,7]. Splenic lymphoid follicles become disorganized with age, and exhibit reduced numbers of small lymphocytes and active germinal centers[8]. However, despite the atrophy of lymphoid organs, T cells isolated from the spleens or lymph nodes of aged mice demonstrated the capacity to produce large amounts of cytokines, including IL-4, IFNc, IL-3, and IL-5 [3,9]. Our results showed that aged tissues expressed very low mRNA levels of IL-2, IL-4, IL-6, IFNc, and TGFb when recalculated per gram of the tissue. This
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suggested that, although aged splenic cells can produce more cytokines than young splenic cells in culture, this does not occur within spleen tissues. Luscieti et al. showed that two out of six lymph nodes showed increases in the relative size of the paracortical zone with advancing age. This suggested that reductions of T cell mass are not the same within different tissue lymph nodes and that the involution of a lymph node does not always indicate a reduction of T cell number [10]. Thus, organ involution is an insufficient explanation for the decline in cytokine expression within tissues. The effects of involutional processes on tissue cytokine production with aging require a more sophisticated explanation. Based on our findings obtained with real-time PCR analysis, the expression of all measured cytokines declined markedly with age in bone marrow. This is in line with findings from Wang et al. [11]. They showed that, during the process of aging, the number of bone marrow macrophages increased, but at the same time, the ability of these cells to produce tumor necrosis factor alpha (TNF a) decreased. TNF a is crucial for stromal production of other cytokines, like IL-6; thus, a reduction in TNF a implies a similar reduction in those cytokines. Our findings confirmed this in terms of gene expression in tissue homogenates. Our findings support the general opinion that immune functionality diminishes with age. In particular, we found that IL-2 mRNA levels declined in lymphoid organs during the aging process. This also supported the previous suggestion that the age-related decline in IL-2 expression occurred at the level of transcription [12]. In conclusion, we found that immunosenescence was accompanied by different patterns of cytokine expression in different
organs. To our knowledge, this is the first study that measured a cytokine transcription profile in rat lymph nodes, spleen, and bone marrow at different stages of life. Because cytokine production serves as a measure of immune system efficiency, elucidating age-related changes in the cytokine profile within lymphoid organs may provide a more precise assessment of the condition of the immune system late in life. References [1] Frasca D, Pucci S, Goso S, Barattini P, Barile S, Pioli C, et al. Regulation of cytokine production in aging: use of recombinant cytokines to upregulated mitogen-stimulated spleen cells. Mech Ageing Dev 1997;93:157–69. [2] Gardner EM, Murasko DM. Age-related changes in type 1 and type 2 cytokine production in humans. Biogerontology 2002;3:271–89. [3] Wakikawa A, Utsuyama M, Wakabayashi A, Kitagawa M, Hirokawa K. Agerelated alterations of cytokine production profile by T cell subsets in mice: a flow cytometric study. Exp Gerontol 1999;34:231–42. [4] PE Applied Biosystems, Sequence detector user bulletin #2; 1997 Dec 11. [5] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCt method. Methods 2001;25:402–8. [6] Albright JW, Mease RC, Lambert C, Albright JF. Effects of aging on the dynamics of lymphocyte organ distribution in mice. Use of a radioiodinated cell membrane probe. Mech Ageing Dev 1998;101(3):197–211. [7] Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing. J Pathol 2007;211(2):144–56. [8] Metcalf D, Moulds R, Pike B. Influence of the spleen and thymus on immune responses in ageing mice. Clin Exp Immunol 1967;2:109–20. [9] Hobbs MV et al. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J Immunol 1993;150:3602–14. [10] Luscieti P, Hubschmid T, Cottier H, Hess MW, Sobin LH. Human lymph node morphology as a function of age and site. J Clin Pathol 1980;33:454–61. [11] Wang CQ, Xiao H, Lipschitz DA. Effect of age on marrow macrophage number and function. Aging 1995;7:379–84. [12] Pahlavani MA, Richardson A. The effect of age on the expression of interleukin2. Mech Ageing Dev 1996;89:125–54.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Diminished cytokines gene expression in lymphoid organs of healthy aged rats _ Maria Pachówka a,⇑, Jolanta Makula b, Grazyna Korczak-Kowalska a,b a b
Department of Immunology, University of Warsaw, Warsaw, Poland Transplantation Institute, Medical University of Warsaw, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 27 April 2010 Received in revised form 1 September 2010 Accepted 9 November 2010 Available online 6 January 2011 Keywords: Aging Cytokine Gene expression Lymphoid tissues Real-time PCR
a b s t r a c t Immunuosenescence-related dysregulation of cytokine production is not fully understood and the roles of cytokines in organ aging have been insufficiently studied. This work aimed to compare the expression of interleukin-2 (IL-2), IL-4, IL-6, interferon-gamma (IFNc), and transforming growth factor-beta (TGFb) in lymphoid organs of young (3 months; n = 10), adult (1 year; n = 10), and aged (2 years; n = 7) rats under healthy, steady-state conditions. Cytokine mRNA levels were determined with TaqMan real-time PCR. In the spleen, all cytokine expression gradually declined with age (for representative cytokine IL-2 averages dCt in spleens of 3 months, 1 year and 2 years rats were 13,645, 13,19 and 15,470, respectively). In lymph nodes, all cytokines except TGF-b showed markedly upregulated expression in adult compared to young rats, but all were expressed at very low levels in aged rats. In bone marrow, adult animals showed enhanced IL-2, IFNc, and TGFb expression, but similar IL-4 and IL-6 expression, compared to young rats. Bone marrow of aged rats showed very low expression of all the measured cytokines. Our results demonstrated that changes occurred unevenly with aging in different immune system compartments. Ó 2011 Published by Elsevier Ltd.
1. Introduction The function of the immune system declines with age. Thus, it follows that the complex cytokine network that orchestrates cell–cell interactions is also affected by age. Age-related changes in cytokine production have been investigated previously in both humans and rodents [1–3]. However, most previous studies were performed with either peripheral blood cells isolated from humans or with lymphoid organ cells isolated from rodents (e.g., spleen or lymph nodes). These isolates were typically cultured in vitro, stimulated with mitogens (antigens or polyclonal activators), and measured based on secreted cytokines in cell supernatants. Due to different culture conditions, stimulation methods, and measurement techniques, variable results have been reported. Therefore, in order to determine the in vivo changes in cytokine production that take place during the aging process, in this study, we used lymphoid tissue homogenates from healthy young, adult, and aged rats. The aim of this study was to study mRNA expression of cytokines in lymphoid organs (lymph nodes, spleen, and bone marrow) under healthy conditions. We studied rats with no vaccinations or immunizations in order to focus on the natural processes that take place during aging. Moreover, we did not isolate lymphocytes, but ⇑ Corresponding author. Address: Miecznikowa 1, 02-096 Warsaw, Poland. Tel.: +48 22 55 41 127; fax: +48 22 55 41 203. E-mail address: [email protected] (M. Pachówka). 1043-4666/$ - see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.cyto.2010.11.007
instead measured changes in cytokine expression in whole tissue homogenates. This may provide clues to in vivo production of cytokines in the context of the local tissue environment. We used TaqMan real-time PCR to analyze the expression of IL2, IFNc, IL-4, IL-6, and TGFb in lymph nodes, spleens, and bone marrow under steady-state conditions in young, adult, and aged rats. We found that each tissue exhibited different patterns of aging. In the period from 3 months to adulthood (1 year old), the lymph node cytokine production, except for TGFb, increased markedly; the spleen cytokine production declined gradually throughout life; and in bone marrow, the IL-2, IFNc, and TGFb production slightly increased with age, but IL-4 and IL-6 production remained stable. In aged rats (2 years old), all tissues exhibited very low numbers of transcripts for all tested cytokines. 2. Materials and methods 2.1. Animals Female Lewis rats were bred in the animal facilities of the Department of Experimental and Clinical Medicine at the Polish Academy of Sciences (Warsaw, Poland). Animals were divided into three groups based on their age. Three-months-old female rats represented the young group (n = 10), 1-year-old female rats represented the adult group (n = 10), and 2-years-old female rats represented the old group (n = 7). The animals were maintained at a stable temperature (23 ± 2 °C) and were provided with an
M. Pachówka et al. / Cytokine 54 (2011) 24–28
unlimited and uninterrupted supply of food and water. Axillary lymph nodes and spleens were isolated and preserved in 70 °C. Bone marrow was isolated from thigh bone and RNA isolation was performed immediately after. All procedures involving animals were reviewed and approved by the First Warsaw Local Ethical Commission for Animal Experimentation (No. 642/2006). All experimental manipulations described herein were performed in strict accordance with the recommendation determined by the ethical commission. 2.2. RNA isolation Lymph nodes, spleens and bone marrows were homogenized in RLT lysis buffer from the RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA was isolated according to the manufacturer’s instructions. 2.3. Reverse transcription Complementary strand DNA was synthesized by combining 2 lg of RNA with 1 ll of 10 mM dNTP and 1 ll of 50 ng/ll random hexamers (Invitrogen Life Technologies) in Eppendorf tubes (Axygen, Union City, USA). The suspension was brought up to a total volume of 10 ll with DEPC-treated water. Tubes were kept for 5 min in a Thermomixer Comfort heat block (Eppendorf, Hamburg, Germany). The tubes were then put on ice. Ten microliters of reaction mixture (Master Mix) was then added to each tube. Each aliquot of reaction mixture consisted of: 2 ll of 10 RT buffer, 2 ll of 0.1 mM DTT, 4 ll of 25 mM MgCl2, 1 ll of RNaseOUT (40 U/ll) and 1 ll of SuperScript III RT (200 U/ll) (Invitrogen Life Technologies). The tubes were kept at 25 °C for 10 min in the heating block. The temperature was then increased to 50 °C, and the tubes were kept in the heating block for another 50 min. For the last 5 min, the temperature was raised to 85 °C. 2.4. Real-time PCR The levels of IL-2, IFN-gamma, IL-4, IL-6, and TGF-beta transcripts were determined by real-time PCR. PCRs were performed in an ABI PRISM 7700 Sequence Detection System. Each of the 27 reaction mixtures contained: template cDNA, TaqMan Universal PCR Master Mix (Applied Biosystems, USA), and TaqMan gene expression assay for the genes of interest (Applied Biosystems, USA). Samples were normalized using rat GAPDH expression. The reaction was carried out under the following conditions: one cycle for 2 min at 50 °C and 10 min at 95 °C; and 40 cycles for 15 s at 95 °C and 1 min at 60 °C. Ct values representing the number of cycles at which the fluorescence value for each sample exceeded the threshold value were recorded. The expression level in each sample was compared with a calibrator. The following formula was applied: gene expression = 2DDCt In brief, relative quantification was performed using 2DDCt method. Reference gene for GAPDH was used. GAPDH as internal control was used for the normalization of the quantity of RNA. For normalization of the results Ct value for GAPDH was subtracted from Ct value for each of target genes (IL-2, IFNc, IL-4, IL-6, TGFb).
DCt ¼ Average CtTarget gene Average CtGAPDH The obtained difference (DCt) was then used to calculate DDCt with the formula:
DDCt ¼ DCt1-year-old=2-years-old rats DCt3-months-old rats The relative gene expression for cytokines normalized to an endogenous reference and relative to a calibrator was expressed as:
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Gene expression level ¼ 2DDCt Relative gene expression for targeted genes in control group (3-months-old rats) equals 1 (since 20 = 1). Relative gene expression for meassured cytokines in lymphoid tissues of 1-year-old and 2-years-old rats was presented as the fold change comparing to the control group [4,5]. 2.5. Statistical analysis All continuous data are expressed as mean ± standard deviation (SD). Analyses were performed to estimate differences using the Turkey test. A p value of <0.05 was considered to be statistically significant. For statistical analysis, only DCt values were used. For better interpretation, data were described in terms of relative gene expression. 3. Results 3.1. Relative gene expression of IL-2 in lymphoid tissues Aged (2-years old) rats exhibited the lowest IL-2 expression levels in lymph nodes, spleen, and bone marrow. In contrast, the relative gene expression of IL-2 was elevated in adult (1-year old) rats in all three examined organs. In particular, in adult rat lymph nodes, IL-2 gene expression was over 12-fold higher than that observed in young (3-months old) rat lymph nodes (Fig. 1). 3.2. Relative gene expression of IL-4 in lymphoid tissues The spleen exhibited a constant decline in IL-4 mRNA levels throughout the aging process. The spleens of aged rats exhibited the lowest relative IL-4 expression. The lymph nodes of adult rats exhibited very high IL-4 gene expression (over 23-fold higher) compared to those of young rats. The lymph nodes of aged rats exhibited significantly lower levels of IL-4 transcripts. The bone marrow of adult rats exhibited a slight increase in IL-4 gene expression compared to the bone marrow of young rats; but the IL-4 levels declined in the bone marrow of aged rats (Fig. 2). 3.3. Relative gene expression of IL-6 in lymphoid tissues Despite the fact that all the examined tissues from aged animals exhibited nearly undetectable IL-6 mRNA levels, the relative IL-6 expression in adult animals differed remarkably among different tissues. The lymph nodes of adult rats exhibited increased IL-6 mRNA levels compared to those of young rats. In contrast, the spleens of adult animals exhibited lower IL-6 mRNA levels compared to those of young rats. Finally, the bone marrow of adult rats exhibited IL-6 expression levels similar to that observed in young rats (Fig. 3). 3.4. Relative gene expression of IFNc in lymphoid tissues The relative gene expression of IFNc showed marked increases between 3 months and 1 year of age. In the lymph nodes and bone marrow, adult rats exhibited, respectively, over 11-fold and over 7-fold higher IFNc expression levels than those observed in young rats. In contrast, in the spleen, IFNc expression levels were similar in adult and young rats. However, with advancing age, significant reductions were observed in the relative expression of IFNc in all three tissues; the lowest level was observed in lymph lodes (Fig. 4).
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Fig. 1. Changes of IL-2 expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IL-2 gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IL-2 in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
Fig. 2. Changes of IL-4 expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IL-4 gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IL-4 in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
Fig. 3. Changes of IL-6 expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IL-6 gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IL-6 in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
3.5. Relative gene expression for TGFb in lymphoid tissues The relative TGFb expression pattern was similar to that observed for IFNc. In lymph nodes and bone marrow, adults rats exhibited in-
creased TGFb expression compared to that observed in young rats. In spleen, TGFb gene expression levels were very similar in young and adult rats. In addition, TGFb expression was significantly reduced in the spleen, lymph nodes, and bone marrow of aged rats (Fig. 5).
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Fig. 4. Changes of IFNc expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of IFNc gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for IFNc in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
Fig. 5. Changes of TGFb expression in lymph nodes, spleens and bone marrows of young (3-months-old), adult (one-year-old) and old (2-years-old) rats. mRNA level was assed by real-time PCR. GAPDH was used for normalization. (A) Expression profiles of TGFb gene expression presented as average DCt values in lymph nodes, spleens and bone marrows obtained from 3-months-old, 1-year-old and 2-years-old rats. ⁄p < 0.05 (B) Relative gene expression for TGFb in lymph nodes of 3-months-old, 1-year-old and 2-years-old rats presented as the fold change (2DDCt method used for defining relative gene expression using control samples as the calibrator).
4. Discussion In this study, we investigated the relative gene expression of five important cytokines, IL-2, IL-4, IL-6, IFNc, and TGFb, in the lymphoid organs of young, adult, and aged rats. We choose lymphoid organs, not peripheral blood mononuclear cells (PBMCs), because it is well known that lymphocytes circulating in blood are not representative for the whole immunological system. Hence, data based on PBMC investigations are not conclusive for the analyzing of the immune system activity. We found that aging affected cytokine mRNA expression differently in lymph nodes, spleen, and bone marrow. From youth, to adulthood, and from adulthood to later life, the spleen exhibited an age-related decline in cytokine production. In contrast, from youth to adulthood, the lymph nodes exhibited marked increases in the expression of all measured cytokines, except TGFb; moreover, in bone marrow, the TGFb, IL-2, and IFNc expression slightly rose and production of IL-4 and IL-6 remained stable. Then, from adulthood to later life, in lymph nodes, the expression of all cytokines dropped to nearly undetectable levels, and in bone marrow, we detected only very low levels of all cytokine mRNAs. Our finding that all aged tissues exhibited an age-related decline in cytokine production was inconsistent with the hypothesis that aging causes a decline in Th1 cytokine production (IL-2 and IFNc) and an increase in Th2 cytokine production (IL-4 and IL-6) [2]. Our results demonstrated that the predominant Th2 response
observed in the blood was not reflective of the organs. The discrepancy between our results and earlier findings might be explained by hypothesizing that aging does not diminish the ability of cells to produce cytokines, but aging diminishes the environmental stimulation. Thus, the mitogen stimulation provided in in vitro experiments is sufficient to induce isolated aged cells to secrete cytokines in cell culture. However, under normal aging conditions within the organ, there are no strong inducers that can stimulate cytokine secretion, either Th1 or Th2 cytokines. Our results suggested that, up to 1 year of age, the microenvironment promotes cytokine secretion in lymphoid organs, like lymph nodes. But, during the 2nd year of age, lymphoid tissues become devoid of cytokine transcripts; however, T cells and other cells retain the capability of cytokine production, as reported previously [3]. It is well known that lymphoid organs exhibit atrophy with aging. Morphological studies on human lymph nodes have revealed numerous age-associated changes, including increased adipose tissue content and a reduction in germinal center development [6,7]. Splenic lymphoid follicles become disorganized with age, and exhibit reduced numbers of small lymphocytes and active germinal centers[8]. However, despite the atrophy of lymphoid organs, T cells isolated from the spleens or lymph nodes of aged mice demonstrated the capacity to produce large amounts of cytokines, including IL-4, IFNc, IL-3, and IL-5 [3,9]. Our results showed that aged tissues expressed very low mRNA levels of IL-2, IL-4, IL-6, IFNc, and TGFb when recalculated per gram of the tissue. This
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suggested that, although aged splenic cells can produce more cytokines than young splenic cells in culture, this does not occur within spleen tissues. Luscieti et al. showed that two out of six lymph nodes showed increases in the relative size of the paracortical zone with advancing age. This suggested that reductions of T cell mass are not the same within different tissue lymph nodes and that the involution of a lymph node does not always indicate a reduction of T cell number [10]. Thus, organ involution is an insufficient explanation for the decline in cytokine expression within tissues. The effects of involutional processes on tissue cytokine production with aging require a more sophisticated explanation. Based on our findings obtained with real-time PCR analysis, the expression of all measured cytokines declined markedly with age in bone marrow. This is in line with findings from Wang et al. [11]. They showed that, during the process of aging, the number of bone marrow macrophages increased, but at the same time, the ability of these cells to produce tumor necrosis factor alpha (TNF a) decreased. TNF a is crucial for stromal production of other cytokines, like IL-6; thus, a reduction in TNF a implies a similar reduction in those cytokines. Our findings confirmed this in terms of gene expression in tissue homogenates. Our findings support the general opinion that immune functionality diminishes with age. In particular, we found that IL-2 mRNA levels declined in lymphoid organs during the aging process. This also supported the previous suggestion that the age-related decline in IL-2 expression occurred at the level of transcription [12]. In conclusion, we found that immunosenescence was accompanied by different patterns of cytokine expression in different
organs. To our knowledge, this is the first study that measured a cytokine transcription profile in rat lymph nodes, spleen, and bone marrow at different stages of life. Because cytokine production serves as a measure of immune system efficiency, elucidating age-related changes in the cytokine profile within lymphoid organs may provide a more precise assessment of the condition of the immune system late in life. References [1] Frasca D, Pucci S, Goso S, Barattini P, Barile S, Pioli C, et al. Regulation of cytokine production in aging: use of recombinant cytokines to upregulated mitogen-stimulated spleen cells. Mech Ageing Dev 1997;93:157–69. [2] Gardner EM, Murasko DM. Age-related changes in type 1 and type 2 cytokine production in humans. Biogerontology 2002;3:271–89. [3] Wakikawa A, Utsuyama M, Wakabayashi A, Kitagawa M, Hirokawa K. Agerelated alterations of cytokine production profile by T cell subsets in mice: a flow cytometric study. Exp Gerontol 1999;34:231–42. [4] PE Applied Biosystems, Sequence detector user bulletin #2; 1997 Dec 11. [5] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2DDCt method. Methods 2001;25:402–8. [6] Albright JW, Mease RC, Lambert C, Albright JF. Effects of aging on the dynamics of lymphocyte organ distribution in mice. Use of a radioiodinated cell membrane probe. Mech Ageing Dev 1998;101(3):197–211. [7] Gruver AL, Hudson LL, Sempowski GD. Immunosenescence of ageing. J Pathol 2007;211(2):144–56. [8] Metcalf D, Moulds R, Pike B. Influence of the spleen and thymus on immune responses in ageing mice. Clin Exp Immunol 1967;2:109–20. [9] Hobbs MV et al. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J Immunol 1993;150:3602–14. [10] Luscieti P, Hubschmid T, Cottier H, Hess MW, Sobin LH. Human lymph node morphology as a function of age and site. J Clin Pathol 1980;33:454–61. [11] Wang CQ, Xiao H, Lipschitz DA. Effect of age on marrow macrophage number and function. Aging 1995;7:379–84. [12] Pahlavani MA, Richardson A. The effect of age on the expression of interleukin2. Mech Ageing Dev 1996;89:125–54.