Experimental Biology and Medicine >
Volume 229, Number 6 >
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* Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo 158–8501, Japan; Department of Anatomy and Developmental Biology, Nihon University School of Medicine, Tokyo 173–8610, Japan; Department of Molecular and Cellular Biology, Faculty of Science, Niigata University, Niigata 950–2181, Japan; and Center for Biological Safety and Research, National Institute of Health Sciences, Tokyo 158–8501, Japan
1 Center for Biological Safety and Research, National Institute of Health Sciences, 1-18-1 Kamiyohga, Setagayaku, Tokyo 158–8501, Japan. E-mail: tohru{at}nihs.go.jp
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Keywords: aging, B-lymphopoiesis, interleukin-7, transforming growth factor-β, , senescence-accelerated mice, homeosuppression
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B lymphopoiesis is suppressed during senescence not only in mice but also in humans. A decrease in interleukin-7 (IL-7) production by stromal cells and a simultaneous reduction in B lymphocyte reactivity to IL-7 are considered as a possible background for this negative senescent regulation. Furthermore, in addition to senescence, B cells were noted to be regulated by two pathways not only for IL-7 but also for transforming growth factor-β (TGF-β) in regular mice; that is, not only the downregulation of the former but also the upregulation of the latter simultaneously play a role in suppression regulation (8). This is in good agreement with the observation that the supplementation of IL-7 could not compensate for the B cell suppression. In this study, possible senescence-associated alterations in the productions of IL-7 and TGF-β are examined in senescence-accelerated mice (SAMs).
Senescence-accelerated mice provide a unique model system for studying senescence or aging in higher organisms, because they exhibit a marked acceleration of aging, which has been confirmed to be the same manner as that observed in the regular mice. Senescence-accelerated mice are characterized by the early onset of aging (mean life span of 40 weeks under conventional conditions), loss of general behavioral activity, increased skin coarseness, and spinal lordokyphosis (9). Because the number of splenic cells starts to decrease at approximately 30 weeks old, the SAMs used were, in general, 30 weeks old or slightly older. Although one must carefully interpret the results of studies using SAMs because the mechanism of "accelerated aging" may not be associated with that of "normal aging," results of previous studies conducted by other researchers and ourselves indicate that SAMs are a suitable model for predicting the possible mechanism of aging in hematopoietic systems (10–14).
The purposes of this study are to confirm the status of B lymphopoiesis in SAMs compared with that in other regular strains and to elucidate the mechanism of age-related changes in B lymphopoiesis in SAMs. Here, we examined age-related changes in the number and function of B cell progenitors in the BM and their supportive microenvironment.
| Materials and Methods |
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Preparation of BM Cells.
The BM cell suspensions were prepared by repeatedly flushing the cells from femurs and dispersing them by trituration through a 23-gauge hypodermic needle with the Iscove-modified Dulbecco medium (IMDM; Invitrogen Corp., Carlsbad, CA) or RPMI 1640 medium (Invitrogen).
In Vitro Colony Assays.
Colony-forming unit pre-B (CFU-pre-B) cells were assayed by suspending mononuclear cells in 1-ml aliquots of the recombinant IL-7 (rIL-7)–supplemented MethoCult M 3630 medium (Stem Cell Technologies Inc., Vancouver, Canada) in 35-mm, plastic petri dishes. Femoral BM cells from three mice per group were pooled and assayed. A MethoCult M 3630 medium consisting of 1 ml of the semisolid IMDM medium containing 1% methylcellulose, 30% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT), 0.1 mM 2-mercaptoethanol (2-ME), 2 mM L-glutamine, and 10 ng/ml of rIL-7 (R&D Systems, Inc., Minneapolis, MN) was used. Granulocyte-macrophage colony-forming units (GM-CFUs) were assayed by suspending mononuclear cells in the alpha medium containing 1% methyl cellulose, 30% FBS, 1% bovine serum albumin, 1 mM 2-ME, and 10 ng/ml of granulocyte-macrophage colony-stimulating factor (Genzyme, Cambridge, MA) and plating 1-ml aliquots in 35-mm, plastic dishes. Both CFU-pre-B cells and GM-CFUs in culture plates in triplicate were incubated at 37oC in a fully humidified atmosphere of 5% carbon dioxide in air. Aggregates of 50 or more cells in 7-day cultures were counted as colonies. Aggregates ranging from 10 to 49 cells were counted as clusters.
Co-culture of Stromal Monolayers and Pro-B/Pre-B Cell-Rich Populations.
Stromal monolayers were prepared by culturing BM cells derived from young or senescent SAMs at 1 x 106/ml in 96-well Coster 3596 or 24-well Falcon 3047 flat-bottomed plates in 0.2 or 1 ml of the RPMI 1640 medium supplemented with 20% FBS. Con-fluent adherent layers were formed after 7 days. To obtain pro-B/pre-B cell-rich populations, the bulk culture of pooled BM cells from young SAMs stimulated with rIL-7 was performed as described previously (4). Briefly, BM cells from young SAMs were cultured at 1 x 106 cells/ml in RPMI 1640 supplemented with 20% FBS, 2 x 10–5 M 2-ME, 1% L-glutamine, and 2 ng/ml of murine rIL-7 (Genzyme) and plated in six-well Coster 3516 culture trays. Nonadherent cells were harvested after 4 days of culture. This bulk culture provided a highly rich (>10-fold) source of IL-7–responsive B220+, CD43+, IgM–, pro-B/pre-B cells (data not shown). Pro-B/pre-B cell-rich populations were suspended at 5 x 104/ml in RPMI 1640 supplemented with 20% FBS, 2 x 10–5 M 2-ME, 1% L-glutamine, and 1 ng/ml of murine rIL-7. Aliquots (0.1 or 1.0 ml) of this cell suspension were added to established stromal cell monolayers in 96- and 24-well flat-bottomed trays, respectively, and co-cultured at 37oC in a fully humidified atmosphere of 5% carbon dioxide in air. Nonadherent cells were harvested after 3 days, counted, and cloned using the CFU-pre-B colony assay system.
Extraction of Total RNA and Polymerase Chain Reaction (PCR).
Total RNA was extracted from BM cells using the TRIsol reagent (Invitrogen) according to the manufacturer’s instructions. First, messenger RNA (mRNA) was reverse transcribed using superscript (Life Technologies, Grand Island, NY) and random hexamers. Next, PCR amplification of complementary DNA (cDNA) was performed with the graded dilution of cDNA for semi-quantitative evaluation of IL-7 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression under the following conditions: IL-7 cDNA, 95 oC for 1 min, 55oC for 2 mins, and 72oC for 3 mins for 35 cycles; and GAPDH cDNA, 94oC for 30 secs, 60oC for 30 secs, and 72oC for 1 min for 20 cycles. Murine IL-7 and GAPDH primers were synthesized based on a published cDNA sequence (15):
IL-7 (sense) 5'-GCCTGTCACATCTGAGTGGC-3'
IL-7 (antisense) 5'-CAGGAGGCATCCAG-GAACTTCTG-3'
GAPDH (sense) 5'-TGAAGGTCGGTGTGAACG-GATTTGGC-3'
GAPDH (antisense) 5'-CATGTAGGCCATGAGGTC-CACCAC-3'
The expected amplified PCR products were 496 and 982 base pairs long for IL-7 and GAPDH, respectively. The PCR products were photographed using the Bio-Rad 2000 gel documentation system (Bio-Rad Laboratories, Hercules, CA), and intensities of expressions were evaluated by ImageGauge version 3.11 (Science Lab 98 for Windows; Fuji Film, Tokyo, Japan). In this experiment, GAPDH expressions were not altered among the experimental groups.
Effect of Anti–TGF-β Antibody on Growth of Pro-B/Pre-B Cell-Rich Population Co-Cultured with Stromal Cells.
To examine the effect of TGF-β produced by stromal cells on the growth of pro-B/pre-B cells, a neutralizing monoclonal antibody (mAb) to TGF-β (mouse IgG1 isotype, R&D Systems) at dilutions ranging from 1–10 µg/ml was added to the co-culture system. The mouse IgG1 isotype (R&D Systems) was used as mock control. The number of nonadherent cells in the co-cultures was determined 3 days later.
Determination of Level of TGF-β Protein Produced by Cultured Stromal Cells.
Stromal monolayers were prepared by culturing BM cells from young and senescent SAMs at 1 x 106/ml in 24-well Falcon 3047 flat-bottomed plates in 1 ml of the RPMI 1640 medium supplemented with 20% FBS. Confluent adherent layers were obtained after 7 days. The supernatant in the culture plates was removed; and then 1 ml of RPMI 1640, supplemented with 20% FBS, 2 x 10–5 M 2-ME, and 1% L-glutamine were added to the culture plates. The culture medium was collected after 7 days of culture and was used for the determination of the level of the TGF-β protein produced by stromal cells. The TGF-β concentration in the culture medium was determined using a TGF-β–specific enzyme-linked immunoabsorbent assay (ELISA) kit (R&D Systems) according to the manufacturer’s instructions. All the samples were assayed in triplicate. The samples were acid activated (16) by adding 1/5 vol of 1 N hydrochloride at room temperature and neutralized after 10 mins by adding of 1/5 vol of 1.2 N NaOH in 0.5 M HEPES, and the mixture was diluted with the same volume of calibrator diluents in the ELISA kit.
Statistical Analysis.
Data were analyzed using the analysis of variance (ANOVA). Values were considered significantly different at P < 0.05.
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Significant Decrease in Number of Large Pre-B Colonies.
Among B cell colonies of various sizes, we noted that the number of relatively larger B cell colonies decreased significantly with aging (Fig. 1). The number of cells per colony ranged from 50–5000. Therefore, CFU-pre-B cell colonies in Table 1 were categorized according to their size, namely, small (50–200 cells), intermediate (201–3000), and large (>3000 cells). As shown in Figure 1, the numbers of large, intermediate, and small B cell colonies for all groups decreased with age (58.3% for large colonies, 75.8% for intermediate colonies, 78.4% for small colonies in 30-week-old mice relative to those in 12-week-old mice; 22.1% for large colonies, 52.0% for intermediate colonies, and 76.5% for small colonies in 36-week-old mice relative to those in 12-week-old mice). The decrease in the numbers was statistically most significant for large colonies in 30-and 36-week-old mice relative to those to 12-week-old control (P < 0.001 and P < 0.005, respectively) and also for intermediate colonies in 36-week-old mice relative to those in 12-week-old control (P < 0.05).
Decrease in IL-7 Expression Level in BM.
As observed in the regular senescent mice, the number of pre-B cell progenitors also decreased in SAMs. Therefore, the expression level of IL-7, which is known to be a pre-B cell stimulator, was evaluated in BM cells. IL-7 expression level decrease during senescence (17–19). In this study, IL-7 mRNA expression level was evaluated by reverse transcriptase (RT)–PCR. The BM stromal cell–derived IL-7 is a positive regulator of in vivo B lymphopoiesis. As shown in Figure 2, the IL-7 mRNA expression level in BM cells from senescent mice was 6.2% that from young mice. These findings are comparable to those in the literature (17–19). Further experiments using SAMs were designed.
Decrease in Proliferative Capacity of B Cell Progenitors, Pre-B Cell Response to IL-7.
The decrease in IL-7 expression level is also associated with the decrease in the responsiveness of pre-B cells to IL-7. To evaluate such responsiveness, we used a recloning assay to determine the proliferative capacity of the progeny of CFU-pre-B cells. Cells derived from 36 large colonies were pooled and recloned for 7 days in a semisolid medium supplemented with rIL-7. Table 2 shows the results of the recloning study. The numbers of secondary colonies, which included small (50–200 cells) and intermediate (201–3000) colonies and clusters (10–49 cells) generated from individual large primary colonies and derived from 30-week-old and 36-week-old femoral BMs, decreased significantly to 69.0% and 2.7%, respectively, that of secondary colonies grown from large primary colonies derived from 12-week-old femoral BM. Furthermore, cells from small primary colonies derived from either young or senescent mouse BM formed no secondary colonies. In B lymphopoiesis, unlike in the case of myelogenous progenitors, the results indicate that the responsiveness of CFU-pre-B cells to IL-7 decreases with age.
Decrease in Maintenance Capacity of Stromal Cells for B Lymphopoiesis.
Although the pre-B progenitor cells were altered during senescence, a decrease in the maintenance capacity of stromal cells for B cell lineages may also be of importance in association with hematopoietic senescence (17, 18, 20). Using a co-culture system in 24-well flat-bottomed trays, we determined whether the capacity of stromal cells to support B lymphopoiesis is altered with age. Interestingly and unexpectedly, the number of lymphocytes recovered from the coculture of pro-B/pre-B cells with young stromal cells was significantly lower than that recovered from senescent stromal cells (Fig. 3A). In contrast, the total number of CFU-pre-B cells recovered from the co-culture with young stromal cells was significantly higher than that recovered from the co-culture with senescent stromal cells (Fig. 3B).
Decrease in TGF-β Production by Senescent Stromal Cells.
On the basis of the above-mentioned co-culture data, we propose a hypothesis that the CFU-pre-B inhibitory activity of BM cells may reside predominantly in young stromal cells rather than in senescent stromal cells. Because stromal cells produce BM-derived TGF-β (21) and are also a negative regulator of B lymphopoiesis (22–24), we investigated whether TGF-β production by stromal cells is reduced with age. Figure 4 shows percent changes in the number of the same seeded BM cells cocultured with young stromal cells (open circles) or senescent stromal cells (closed circles) in 96-well flat-bottomed trays after adding a graded dose of a neutralizing mAb to TGF-β. The high-dose group (10 µg/ml and more; data not shown) exhibited a toxic effect, but the lower-dose group showed a significant difference of responses between the young and senescent groups, implying that the proliferation of the senescent group was unexpectedly not recovered by the neutralizing antibody, whereas (although it was not expected) the proliferation of the young group was prominently recovered at 1- and 5-µg/ml doses (statistical significance, P < 0.001 in the group of 5 µg/ml). Thus, despite the prominent decrease in IL-7 expression level and in the biological activity of IL-7 in the BM, TGF-β production seemed to have unexpectedly decreased in the senescent group. To confirm the decrease in TGF-β production by stromal cells with age, we directly measured TGF-β protein level in the supernatant of cultured stromal cells derived from young and senescent SAMs using ELISA. Figure 5 shows that the TGF-β protein level in the supernatant of cultured senescent stromal cells is markedly lower than that of cultured young stroma, thus implying that TGF-β production by stromal cells decreases with age (634.0 ± 36.0 vs. 337.0 ± 37.9, young and old, respectively, P < 0.001).
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The expression level of IL-7, a pre-B cell stimulator, was evaluated in BM cells, because IL-7 production decreases during senescence (17–19). Mice in which the IL-7 gene has been knocked out manifest a prominent decrease in the number of pre-B lymphocytes and a severe impairment in capacity for self-renewal in the pre-B cell compartment, even though the number of pro-B cells appears to be normal. Phenotypically, age-related changes seem closer to the changes in the B lymphopoiesis observed in aged mice (27). Our data showed that the IL-7 mRNA expression level in freshly isolated BM cells decreases with age (Fig. 2). We attempted to measure IL-7 expression level only in freshly isolated BM cells and BM stromal cells, because the expression of IL-7 is easily upregulated immediately after the start of culture. Because of the limited materials for ELISA, protein expression level was not determined. These findings are consistent with those of Updyke et al. (17), who reported that the relative quantity of the IL-7 protein released into the medium for long-term B cell culture decreases with age. A study by Stephan et al. (18) suggested that the age-related decrease in the function of BM cells is associated with the impaired release of IL-7. Interestingly, a novel mutant mouse model of aging, klotho, was reported to exhibit a similar significant decrease in the level of IL-7 gene expression in freshly isolated BM cells as determined by RT-PCR analysis (19); the mouse initially exhibits multiple disorders that resemble various aging phenotypes. Based on the findings by other researchers and ourselves, it seems likely that IL-7 production by BM stromal cells decreases with age. As seen in Figures 1–4, comparable results were obtained in reports available in the literature; hence, the present findings obtained through the experiment using SAMs may be applicable to the analysis of natural aging in regular mice.
Next, we demonstrated that the production of TGF-β by marrow stromal cells decreased with age, although the mechanism underlying this phenomenon is not yet known. Young stromal cells inhibited B cell proliferation in the co-culture system, and this inhibition was reversed by treatment with antibodies to TGF-β. The results of the co-culture system demonstrated that significantly fewer lymphocytes could be recovered from the co-culture system with young stromal cells than with senescent stromal cells; conversely, a significantly higher number of CFU-pre-B cells is maintained in the co-culture system with young stromal cells than with senescent stromal cells (Fig. 3A and B). Moreover, the neutralizing antibody to TGF-β restored the proliferative capacity of pro-B/pre-B cells co-cultured with young stromal cells but not that of those co-cultured with senescent stromal cells (Fig. 4). Furthermore, the TGF-β protein level in supernatant of cultured senescent stromal cells is markedly lower than that of young stromal cells (Fig. 5). These results imply that senescent stromal cells are not capable of producing TGF-β. These data agree with those reported by Dubinett et al. (28) that IL-7 downregulates both mRNA expression and protein production of TGF-β by murine macrophages. Thus, it seems unlikely that exogenous IL-7 added to our co-culture system would induce TGF-β production by stromal cells derived from a young stroma. Furthermore, Gazit et al. (29) have recently reported that fibroblast CFU (CFU-F) isolated from senescent mice produces less TGF-β in vitro than CFU-F from young mice and that the matrix of long bones of senescent mice contains less TGF-β than that of young mice. These data suggest that the production of the CFU-pre-B cell regulator TGF-β by stromal cells may decrease with age. Consequently, CFU-pre-B cells co-cultured with senescent stromal cells may proliferate and/or differentiate more rapidly than CFU-pre-B cells co-cultured with young stromal cells in the presence of IL-7.
In the present study, we observed that the CFU-pre-B cell number in the BM decreased with age, whereas, as we have observed previously (13), the total number of splenic B cells remained relatively unchanged. These findings are consistent with those observed in other murine strains by other researchers (3–6) and have been considered to be mediated by a decrease in B lymphocyte production in the BM and increased longevity of mature B cells (30). Furthermore, our data revealed an intrinsic defect in the B-progenitor-cell response to IL-7, as well as an age-related impaired production of not only IL-7 but also TGF-β by stromal cells. In SAMs, the arrest of pro-B cell maturation with advanced aging was evidently associated with the decrease in the number of pre-B cells. This may be explained by the coexistence of an intrinsic defect in the B-progenitor-cell response to IL-7 (i.e., pre-B cells and more immature pro-B cells); this interpretation is in good agreement with previous reports (6). Moreover, a decrease in IL-7 production by stromal cells during aging was confirmed (Fig. 2), which is evident in regular mice (17, 18).
The present study revealed that senescent B lympho-poiesis is suppressed, the background mechanism of which is unlikely different from mechanical B cell damage and its acute responses. Although B cell damage may be based on a positive circuit (i.e., an increase in IL-7 production associated with a decrease in TGF-β production) (28, 31, 32), our present data clearly show that TGF-β production is rather suppressed despite the prominent decrease in IL-7 production. Such homeostatic B lymphopoiesis balanced at the lower level may be a prominent characteristic of the basic mechanism of B lymphopoietic senescence, although the details of this mechanism are as yet unknown.
Another objective of our current study is to address the issue of using SAMs as an experimental mouse model for predicting the possible basic mechanism of senescence and B lymphopoiesis during aging. Aging is a physiological process and is likely controlled by a combination of many different factors. Whether the determinant of accelerated aging in SAMs is the same as that of normal aging in mice remains to be elucidated. However, the determinant factors for aging of an organism are, at present, poorly understood. Thus, different experimental approaches using animal models such as SAMs may provide an insight into such factors, because the study of abnormal systems has often led to the clarification of how a normal system functions.
Our present study, performed using SAMs and focusing on the quantity and quality of B lymphopoietic progenitor cells, suggests age-related alterations in lymphopoietic progenitor cells. Among them, the changes shown in Tables 1 and 2 and Figure 1 are essentially identical to those observed in regular mouse strains as previously reported. Namely, the decreased IL-7 responsiveness of BM cells from aged mice appears to be associated with both the decrease in the number of IL-7–responsive cells and the decrease in colony size and to correlate with findings in other strains (3–6). Furthermore, the number of secondary colonies generated by the progeny of CFU-pre-B cells derived from large primary colonies was significantly smaller for the BM of senescent mice than for that of young mice (Table 2). Note that there seems to be an almost complete arrest in the production of secondary CFU-pre-B colonies from 36-week-old mice (Table 2), whereas comparable primary BM cells produced the same amount of GM-CFU (109%) from 12-week-old mice, and 65.0% of CFU-pre-B colonies was maintained (Table 1) in 12-week-old mice. The decrease in the number of secondary colonies was prominently observed in the most senescent age group (i.e., 36 weeks old) at the level of "cluster," because there seems to be a split between the time to senescence for spleen atrophy and that for substantial hemopoietic arrest in in vitro colonization. Stephan et al. (25) reported that a small percentage of BM pro-B cells from aged mice undergo cell cycle and that a large percentage of these cells enter G0/G1 after stimulation with IL-7, suggesting an impairment or delay in their ability to undergo cell division after IL-7 stimulation. The surrogate light chain is a component of the pre-B cell receptor, which is critical for Ig-variable heavy chain selection, cellular proliferation, and survival in the pre-B stage. Sherwood et al. (26) and Frasca et al. (33) reported that surrogate L chain mRNA and protein levels in IL-7–expanded B cell precursors decrease with age, which is associated with decreased protein levels of the E2A-encoded transcription factors, E47 and E12 (33). Based on these results, impairment in the IL-7 receptor function and its signal transduction in pro-B/pre-B cells may underlie the decrease in B cell production with age. It seems likely that the reduced generation of secondary colonies may be due in part to the deterioration of the proliferative capacity of B-cell progenitors from senescent mice in response to IL-7 rather than to the exclusive differentiation of B-cell progenitors to mature B cells in response to IL-7. In this regard, senescent changes observed in B lymphopoiesis in SAMs may be assumed to be identical to those reported in regular mice.
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| Acknowledgments |
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Received for publication August 1, 2003. Accepted for publication March 1, 2004.
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