CD27+ (memory) B cell decrease and apoptosis-resistant CD27 (naive) B cell increase in aged humans: implications for age-related peripheral B cell developmental disturbances

Yong Chong1, Hideyuki Ikematsu2, Kouzaburo Yamaji1, Mika Nishimura2, Shigeki Nabeshima1, Seizaburo Kashiwagi3 and Jun Hayashi1,4

1 Department of General Medicine, Kyushu University Hospital, Fukuoka, Japan
2 Department of Clinical Research, Hara-Doi Hospital, 6-40-8 Aoba, Higashi-ku, Fukuoka 813-8588, Japan
3 Fukuoka Red Cross Blood Center, Fukuoka, Japan
4 Department of Environmental Medicine and Infectious Diseases, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

Correspondence to: H. Ikematsu; E-mail: ikematsu{at}gray.plala.or.jp


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
To investigate age-related alterations in human humoral immunity, we analyzed the quantity and quality of peripheral B cell subsets, CD27-negative (CD27) and CD27-positive (CD27+) B cells, by flow cytometry analysis in 54 aged individuals (mean age ± SE, 74.6 ± 0.7 years) and 30 young individuals (mean age ± SE, 26.1 ± 0.5 years). CD27 and CD27+ B cells are regarded as naive and memory B cells, respectively. CD38, Ki-67, CD95 and bcl-2 were used as activation, proliferation and apoptotic markers. Susceptibility to apoptosis was evaluated by cell size and annexin-V binding in culture cells. The percentage of CD27+ B cells was significantly lower in aged (mean, 19.2%) individuals than that in young individuals (mean, 28.2%). The opposite was true for CD27 B cells (mean, 80.8% in aged and 71.8% in young) (P < 0.01). The absolute number of CD27+ B cells in aged individuals was significantly less than the number of CD27 B cells. The CD27+ B cells from aged individuals showed little susceptibility to apoptosis, although CD95 expression on the CD27+ B cells was significantly higher in the aged individuals than in the young individuals (P < 0.05). The CD38 and bcl-2 expression on the CD27 B cells was significantly higher in the aged individuals than in the young individuals (P < 0.05). In addition, the CD27 B cells from the aged individuals showed a decreased susceptibility to apoptosis compared with that of the young individuals. These findings suggested that human aging leads to both quantitative and qualitative alterations in the peripheral B cell developmental system, including memory and naive B cell balance and their surface phenotypes.

Keywords: aging, apoptosis, B cells, memory, naive


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Susceptibility to infectious diseases has a serious effect on human longevity, with mortality from infectious diseases, particularly pneumonia, increasing in aged humans (1). One cause for this phenomenon is ‘immunosenescence,’ alteration of host defense mechanisms elicited by aging. The study of immunosenescence in humans is less developed than that in mice. In particular, humoral immunity, which is a defense system against infectious agents, has not yet been fully investigated in aged humans. The prevalence of human peripheral B cell neoplasms, such as B cell-chronic lymphocytic leukemia and B cell lymphoma, rapidly increases with age (2, 3). We have recently demonstrated that somatic mutations of Ig variable region genes accumulate in IgG B cells from aged humans (4). These findings suggest that human aging could affect peripheral B cell development, resulting in augmented B cell oncogenesis.

CD27 antigen expression on B cells primed by antigenic stimulation is important to promote the differentiation of B cells through T–B interaction (5, 6). Few CD27-negative (CD27) B cells carry somatic mutations in Ig variable region genes, whereas CD27-positive (CD27+) B cells accumulate substantial numbers of somatic mutations (7). Accordingly, CD27 and CD27+ B cells are regarded as naive and memory B cells, respectively (8). CD27 (naive) B cells are exclusively produced at birth and, afterwards, by the adolescent period, CD27+ (memory) B cells gradually increase (9). However, little is known about how the composition of these peripheral B cell subsets is altered from the young adult period to that of old age.

Understanding how peripheral B cells are influenced by aging is important for clarifying one aspect of humoral immunity in aged humans. In particular, the analysis of B cell subsets, CD27 and CD27+ B cells, is critical in addressing the alteration of the peripheral B cell developmental process caused by aging. In the present study, the peripheral levels of all B cells and B cell subsets were examined, and their biological characteristics, including activation, proliferation and susceptibility to apoptosis, were investigated in young and aged adults using cell surface and intracellular markers.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Individuals
With informed consent, 30 young individuals (15 men and 15 women) and 54 aged individuals (28 men and 26 women) were the subjects used in the present study. The ages of the young subjects ranged from 22 to 34 years, with a mean age ± SE of 26.1 ± 0.5 years. The ages of the aged subjects ranged from 68 to 87 years, with a mean age ± SE of 74.6 ± 0.7 years. All subjects were independent, were not hospitalized and were not taking any prescription medications. None of the subjects had acute infections. No chronic viral infections, including HIV-1, human T-cell leukemia virus type 1, hepatitis B virus (HBV) and hepatitis C virus (HCV), were detected in any subject. No monoclonal gammopathy was found. Blood sampling was done from March to October 2003.

Measurement of serum Ig
Serum {gamma}-globulin, IgG, IgA, IgM and IgE levels were determined by turbidimetric immunoassay or fluorescence-enzyme immunoassay at Mitsubishi Kagaku Bio-Clinical Laboratories (Tokyo, Japan).

Flow cytometry
For phenotypic analysis of the peripheral CD19-positive cells (B cells), one- to three-color flow cytometry was done using the following FITC-, PE-, or PE–cyanin 5.1 (PC5)-conjugated mouse anti-human mAbs: CD3–PC5, CD19–PC5, CD38–FITC, CD95 (Fas)–FITC (Immunotech, Marseille Cedex, France), CD27–PE (Becton Dickinson, Bridgeport, NJ, USA), Ki-67–FITC, bcl-2–FITC (PharMingen, San Diego, CA, USA) and IgD–FITC (Southern Biotechnology Associates, Birmingham, AL). Ig isotype-matched FITC-, PE- or PC5-conjugated mouse antibodies were used as negative controls for non-specific staining. A 50-µl volume of whole blood was incubated with mAbs, lysed in 1 ml of IO Test 3 Lysing Solution (Immunotech) and prepared for analysis. This analysis was done with the following combinations of conjugated mAbs: CD19–PC5 and CD27–PE; CD19–PC5, CD27–PE and CD38–FITC, and CD19–PC5, CD27–PE and CD95 (Fas)–FITC.

PBMC were isolated from heparinized venous blood by density centrifugation. After the addition of goat serum to block Fc receptors, freshly isolated PBMC were stained with CD19–PC5, CD27–PE and IgD–FITC, and prepared for analysis.

Fixation, permeabilization and intracellular staining of PBMC were performed with the CytoStain Kit (PharMingen) according to the manufacturer's instructions. Briefly, freshly isolated PBMC were stained with CD19–PC5 and CD27–PE. After staining with mAbs, the cells were fixed, permeabilized and stained with Ki-67–FITC or bcl-2–FITC mAbs for three-color flow cytometric analysis.

The stained cells were analyzed by a flow cytometer, Cytoron Absolute (Ortho Diagnostic Systems, Raritan, NJ, USA), using ImmunoCount 2 software (Ortho Diagnostic Systems). Lymphocyte gating was performed using forward- and side-scatter parameters; up to 30 000 cells were acquired from this gate. CD19-positive cells from the lymphogate were used for each analysis.

Measurement of apoptosis
A total of 2.5 x 105 PBMC per well were cultured in 24-well plates for 24 and 48 h in 1 ml of RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 U ml–1 penicillin and 50 µg ml–1 streptomycin, all supplied by GIBCO BRL (Life Technologies, Inc., Gaithersburg, MD, USA). The apoptosis levels of the B cell subsets were evaluated by two markers, small cell size and annexin-V binding, as described elsewhere (1012). Briefly, cultured PBMC were washed, suspended and stained with CD19–PC5 and CD27–PE. After being washed, the cells were re-suspended with 500 µl of binding buffer and stained with annexin-V–FITC for 10 min on ice using the Annexin V–FITC Kit (Immunotech). The cell size of the B cell subsets was measured by the level of forward light scatter using cultured PBMC stained with CD19–PC5 and CD27–PE. Two- and three-color flow cytometry were performed after the above procedures.

Statistical analysis
The Mann–Whitney U-test was performed to compare differences in the analysis data between the young and aged subjects. The association between two related variables was analyzed by using Spearman's rank correlation test.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immunological characteristics in young and aged individuals
Table 1 summarizes the immunological characteristics of the subjects examined in the present study. The absolute number of white blood cells was significantly lower in the aged subjects than in the young subjects (P < 0.01). The absolute number of lymphocytes and CD3 cells was also lower in the aged subjects than in the young subjects (P < 0.01). Serum {gamma}-globulin levels between the young and aged were comparable. The serum mean IgG and IgA levels of the aged subjects were higher than those of the young subjects, although the differences were not significant. The serum IgM levels were significantly lower in the aged subjects than in the young subjects (P < 0.01).


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Table 1. Immunological characteristics in young and aged individuals

 
Circulating CD27 (naive) and CD27+ (memory) B cells in young and aged individuals
Representative FACS analysis of PBMC stained by anti-CD19 and CD27 antibodies is shown in Fig. 1(a). The CD27 B cells were distinctively separate from the CD27+ B cells. The percentage of circulating B cells in lymphocytes was lower in the aged subjects (mean, 10.9%) than in the young subjects (mean, 12.9%) (P < 0.05, Fig. 1b). The absolute number of B cells was also lower in the aged subjects than in the young subjects (P < 0.01, Fig. 1b). The percentage of circulating CD27 B cells of all B cells was significantly higher in the aged subjects (mean, 80.8%) than in the young subjects (mean, 71.8%) (P < 0.01, Fig. 1c). In contrast, the percentage of CD27+ B cell was significantly lower in the aged subjects (mean 19.2%) than in the young subjects (mean, 28.2%) (P < 0.01, Fig. 1d). Although the absolute number of both the CD27 and CD27+ B cells was lower in the aged subject than that in the young subjects (Fig. 1c and d), the rate of reduction in the absolute number was significantly higher in the CD27+ B cells than that in the CD27 B cells (P < 0.01, Fig. 1e). CD27 and CD27+ B cells have been reported as being naive IgD-positive (IgD+) B cells and memory B cells, respectively (13). The percentage of circulating CD27 B cells positively correlates with the percentage of circulating IgD+ CD27 B cells (P < 0.01, r = 0.7), suggesting that the increased CD27 B cell and decreased CD27+ B cell percentages found in the aged subjects were not a result of CD27 down-regulation on memory B cells.



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Fig. 1. (a) Representative FACS analysis of anti-CD19 and anti-CD27 stained PBMC in one young and one aged subject. Numbers in the upper and lower right quadrants show CD27+ and CD27 B cells as a percentage of all peripheral B cells, respectively. (b) Percentage of peripheral CD19+ cells in lymphocytes (left) and absolute number of peripheral CD19+ cells (right) in young and aged subjects. (c) Percentage of peripheral CD27 B cells of all B cells (left) and absolute number of peripheral CD27 B cells (right) in young and aged subjects. (d) Percentage of peripheral CD27+ B cells of all B cells (left) and absolute number of peripheral CD27+ B cells (right) in young and aged subjects. (e) Ratio of absolute number of CD27+ B cells to CD27 B cells in young and aged subjects. The horizontal and vertical bars represent the mean levels and SE, respectively.

 
CD38, CD95 and bcl-2 expression on circulating CD27 (naive) and CD27+ (memory) B cells in young and aged individuals
To investigate the biological characteristics of B cells in aged individuals, activation, proliferation and apoptotic markers were compared between the young and aged subjects. CD38 and Ki-67 expressions on lymphocytes are known to be activation and proliferation markers, respectively (1416). CD95 and bcl-2 are often used as representatives of apoptotic and anti-apoptotic agents expressed by lymphocytes (17, 18).

The percentage of CD38-positive (CD38+) B cells of all B cells was significantly higher in the aged subjects than that in the young subjects (P < 0.05, Table 2). CD38 expression was primarily observed in the CD27 B cells rather than in the CD27+ B cells. The percentage of CD38+ CD27 B cells of all CD27 B cells was significantly higher in the aged subjects than that in the young subjects (P < 0.05, Table 2). The intensity of CD38 on the CD38+ CD27 B cells between the young and aged subjects was comparable. In contrast, CD38 expression on the CD27+ B cells was similarly low in both the young and aged subjects (Table 2). To examine whether the elevated CD38 expression on the B cells from the aged subjects could be explained by B cell proliferation, B cell proliferation was evaluated by Ki-67 expression. The percentages of proliferative B cells between the young subjects (mean, 2.9%) and aged subjects (mean, 2.5%) were comparable. No increased proliferation of either the CD27 or the CD27+ B cells was observed (data not shown).


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Table 2. CD38, CD95 and bcl-2 expression in B cell subsets from young and aged individuals

 
The percentage of CD95-positive (CD95+) B cells of all B cells appeared to be higher in the aged subjects than that in the young subjects, although the difference was not significant (P = 0.15, Table 2). CD95 expression was dominant on the CD27+ B cells but was scarcely observed on the CD27 B cells. The percentage of CD95+ CD27+ B cells of all CD27+ B cells was significantly higher in the aged subjects than in the young subjects (P < 0.05, Table 2). The intensity of CD95 on the CD95+ CD27+ B cells between the young and aged subjects was comparable. In contrast, CD95 expression on the CD27 B cells was low and comparable between the young and aged subjects (Table 2).

Bcl-2 was expressed on almost all of the circulating B cells of the subjects. The intensity of bcl-2 on the B cells was significantly higher in the aged subjects than that in the young subjects (P < 0.05, Table 2). Bcl-2 intensity on the CD27 B cells was significantly higher in the aged subjects than that in the young subjects (P < 0.05, Table 2). Bcl-2 intensity on the CD27+ B cells was higher than that on the CD27 B cells (Table 2). Bcl-2 intensity on the CD27+ B cells appeared to be higher in the aged subjects than that in the young subjects, but the difference was not significant (Table 2).

Susceptibility to apoptosis of circulating CD27 (naive) and CD27+ (memory) B cells in young and aged individuals
To investigate whether altered CD95 and bcl-2 expression on the B cells from aged individuals affect susceptibility to B cell apoptosis, cell size and annexin-V binding in cultured B cells were examined as apoptotic markers. Representative FACS analysis of the cultured B cells is shown as the detection of apoptotic cells in Fig. 2(a). In the analysis of cell size, B cells with small cell size (small B cells) were observed only in the CD27 B cell compartment and were distinctively separate from the cells with normal cell size. Similarly, annexin-V-binding B cells were observed in the CD27 B cell compartment and few were found in the CD27+ B cell compartment. A decrease in CD27 antibody-binding capacity could occur in the apoptotic cells. Thus, it is possible that some apoptotic B cells detected in the present study could come from CD27-positive cells. In the present study, the percentages of CD27 and CD27+ B cells as a percentage of all B cells, 71.8 versus 28.2% in the young subjects and 80.8 versus 19.2% in the aged subjects, were stable in culture at 0, 24 and 48 h. There were no statistical differences in the percentage of CD27+ B cells in the comparison of 0, 24 and 48 h of culture time. If a part of apoptotic B cells detected in the CD27 B cell compartment come from CD27+ B cells, the percentage of CD27+ B cells should decrease with time. The apoptotic B cells were probably derived from the CD27 B cells, and any contribution of the CD27+ B cells to the apoptotic cells would be slight.



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Fig. 2. Susceptibility to apoptosis of peripheral CD27 and CD27+ B cells. Apoptotic B cells were measured using cultured PBMC stained with anti-CD19, anti-CD27 and annexin-V at 24 and 48 h. The level of apoptosis was evaluated by small cell size and annexin-V binding. (a) Representative FACS analysis of B cells with small cell size (small B cells) in one young (right) and one aged (left) subject (top). PBMC were cultured for 24 and 48 h, followed by a dual staining with anti-CD19 and anti-CD27, after which, among gated B cells, the cell sizes of the CD27 and CD27+ B cells were measured by the level of forward light scatter. The small B cells were clearly separate from the B cells with normal cell size. Representative FACS analysis of annexin-V-binding B cells in one young (right) and one aged (left) subject (bottom). PBMC were cultured for 24 and 48 h, followed by a staining with anti-CD19, anti-CD27 and annexin-V, after which annexin-V binding the CD27 and CD27+ B cells were measured among the gated B cells. Culture data at 48 h is shown. Each number in the quadrant is the percentage of all B cells. (b) Percentage of small CD27 B cells of all B cells (left) and percentage of small CD27+ B cells of all B cells (right) in the young and aged subjects. The horizontal and vertical bars represent the mean levels and SE, respectively. (c) Percentage of annexin-V-binding CD27 B cells of all B cells (left) and percentage of annexin-V-binding CD27+ B cells of all B cells (right) in the young and aged subjects. The horizontal and vertical bars represent the mean levels and SE, respectively. (d) Correlation between the percentage of annexin-V-binding CD27 B cells and bcl-2 intensity on CD27 B cells in young (right) and aged (left) subjects.

 
The percentage of small CD27 B cells of all B cells was significantly lower in the aged subjects than that in the young subjects (P < 0.01, Fig. 2b). No small CD27+ B cells were detected in any of the subjects (Fig. 2b). The percentage of annexin-V-binding CD27 B cells of all B cells was significantly lower in the aged subjects than that in the young subjects (P < 0.01, Fig. 2c). Few annexin-V-binding CD27+ B cells were detected in any of the subjects (Fig. 2c). In both the young and aged subjects, the percentage of annexin-V-binding CD27 B cells was inversely correlated with bcl-2 intensity on the CD27 B cells (Fig. 2d). No relationship was found between the percentage of annexin-V-binding CD27 B cells and the percentage of the CD95+ CD27 B cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, both quantitative and qualitative alterations of B cells from aged humans were found, clarifying the characteristics of B cells from aged humans by the analysis of B cell subsets, CD27 and CD27+. In aged humans, the percentage of CD27+ (memory) B cells was dramatically less than the percentage of CD27 B cells. CD27 (naive) B cells exhibited a reduced susceptibility to apoptosis. Interestingly, the serum Ig of aged humans were maintained at a level comparable to those of young humans.

The quantitative and qualitative features of memory B cells in aged humans have not been fully investigated. An extreme depletion of CD27+ (memory) B cells from aged individuals was indicated in the present study (Fig. 1d). Although the absolute number of CD27 (naive) B cells from aged individuals was also reduced, the rate of reduction of the CD27+ B cells was much higher than that of the CD27 B cells (Fig. 1e). Thus, depletion of memory B cells is a characteristic phenomenon in aged humans.

Germinal center B cells display high CD95 expression. Thereafter, most of these B cells are induced into apoptosis (17), which indicates that CD95 antigen expression on B cells might be induced through antigen-specific immune responses. In the analysis of peripheral B cells from healthy individuals, CD38IgD (memory) B cells have been shown to have a significantly higher fraction of CD95 expression than that of CD38+IgD+ (naive) B cells (19). Thus, the induction of CD95-related apoptosis is possibly important as a mechanism of cell death of peripheral memory B cells (post-germinal center B cells). Similar results were obtained in the present study. In addition, aged individuals showed an increased percentage of CD95+ CD27+ B cells compared with that of young individuals (Table 2), suggesting that an apoptotic pathway induced by CD95 signaling is important for memory B cell survival in both aged and young humans.

Bcl-2 intensity, a critical anti-apoptotic factor, was significantly higher in CD27+ B cells than in CD27 B cells (Table 2). In both young and aged individuals, the percentages of CD27+ B cells were stable during culture, and few small or annexin-V-binding CD27+ B cells were detected (Fig. 2b and c). These findings suggest that memory B cells are less susceptible to apoptosis than are naive B cells. The present study does not clarify the mechanism of memory B cell depletion caused by aging. To examine whether an age-related increase of CD95+ CD27+ B cell fractions is related to this loss of memory B cells, further investigation, such as a functional analysis, will be necessary.

Quantitative and qualitative alterations were found in naive B cells as well as in memory B cells. CD27 (naive) B cells became more predominant in the peripheral B cells of aged individuals (Fig. 1c). CD27 B cells of aged individuals showed a decreased susceptibility to apoptosis under the in vitro culture condition, as indicated by cell size and annexin-V binding (Fig. 2b and c). Thus, CD27 B cells seem to become ‘apoptosis-resistant’ in aged humans, as the in vitro condition itself is generally thought to cause cells to become more apoptotic when compared with the in vivo condition. Under the ex vivo condition before culture, CD27 B cells showed a significantly higher bcl-2 intensity in aged individuals than in young individuals (Table 2). Bcl-2 plays a critical role in controlling the apoptotic pathway, and over-expression of bcl-2 is known to increase the resistance of lymphocytes to apoptosis (18). It is possible that the bcl-2-related anti-apoptotic pathway plays an important role in ‘apoptosis resistance’ in the naive B cells of aged humans.

Activated and proliferated B cells augment CD38 expression under various stimulations including T-cell-independent and -dependent responses (14, 20). In general, lymphocyte activation is closely related to increased susceptibility to apoptosis (21). In HIV-infected patients, CD27 B cells with high CD38 intensity have shown a low level of bcl-2 intensity and a high susceptibility to apoptosis (22), suggesting that persistent HIV infection can induce naive B cells to change into an activated and apoptosis-susceptible phenotype. In the present study, CD27 B cells from aged individuals showed an increased percentage of CD38 expression compared with that of young individuals (Table 2), but the intensity was similar between the young and aged individuals. In addition, the CD27 B cells were less susceptible to apoptosis in aged individuals. Although CD38 expression on CD27 B cells increases in frequency with age, it is probable that naive B cells from aged humans are not an activated phenotype.

Some investigators have reported that serum Ig levels of aged humans are significantly higher than those of young humans (23, 24). In the present study, the serum Ig levels of young and aged individuals were comparable (Table 1), suggesting that the production of Ig in aged humans is maintained at a level equivalent to that of young humans. The mechanism of serum Ig maintenance is unknown. Antibody-secreting cells are thought to be derived from peripheral B cells. The age-related B cell alterations found in the present study, as characterized by memory B cell depletion and apoptosis resistance in naive B cells, might affect the differentiation of peripheral B cells into antibody-secreting cells. Further investigation will be necessary to clarify this issue.

Human aging induces both quantitative and qualitative changes in peripheral B cells. During aging, memory B cells decrease and apoptosis-resistant naive B cells increase. We have also reported a higher accumulation of somatic mutations in Ig variable region genes of peripheral IgG B cells among aged humans (4). Thus, it is possible that human aging affects the developmental system of peripheral B cells. This behavior is possibly associated with presumed immunological dysfunctions and hematological disorders in aged humans. The detailed mechanisms and pathological significance of the age-related B cell alterations found in the present study should be addressed.


    Abbreviations
 
PC5   PE–cyanin 5.1

    Notes
 
Transmitting editor: T. Watanabe

Received 25 August 2004, accepted 12 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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