Increased susceptibility of cord blood B lymphocytes to undergo spontaneous apoptosis

Abstract

In this study, we compared the rate of spontaneous apoptosis of B cells from umbilical cord blood with adult B cells and assessed the role of Bcl-2, CD5, interleukin (IL)-4 and B cell-activating factor in B cell spontaneous apoptosis. We found that spontaneous apoptosis of cultured B cells, as assessed by utilizing annexin-V binding, was significantly higher in cord blood than in healthy adult individuals (77·5; 95 CI, 73·5–81·5 versus 59·2; 95 CI, 54–64, respectively, P < 0·0001) and further confirmed by 4′ 6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining. Whereas the expression of B cell-activating factor from the tumour necrosis factor family (BAFF) receptor mRNA was similar in B cells from adults and cord blood, we detected lower levels of circulating BAFF in the serum of cord blood (0·68 ± 0·13 ng versus 1·83 ± 0·54 ng, P = 0·01). The latter may explain, in part, our observation of lower levels of mean fluorescence intensity of Bcl-2 in cord B cells compared with adults (1·6 ± 0·9 versus 2·85 ± 1·3, P = 0·033). CD19⁺ CD5⁺ B cells from cord blood underwent a lower rate of apoptosis in comparison to CD19⁺ CD5⁻ B cells (25·1 ± 9·3% versus 58·5 ± 12·5%, P < 0·0001). This pattern of sensitivity was comparable in adult blood (15 ± 5·5% versus 22·7 ± 9·3%, P = 0·01). Nevertheless, the rate of apoptosis was higher in CD19⁺ CD5⁺ from cord blood compared to CD19⁺ CD5⁺ from adults (25·1 ± 9·3% versus 15 ± 5·5%, P = 0·0013). The addition of rIL-4 (10 u/ml) to cultured cells decreased B cell apoptosis in a similar fashion in both cord and adults blood. This rescue was strengthened when BAFF (100 µg/ml) was further added. Thus, alterations in Bcl-2 or serum BAFF level may explain the increased rate of cord blood B cell apoptosis.

Introduction

Programmed cell death (PCD) is a cellular suicide mechanism that plays a crucial role in homeostasis and in the maintenance of self-tolerance. Alterations in the sensitivity of peripheral blood immune cells (i.e. lymphocytes, monocytes, neutrophils) to undergo apoptosis and the rate of these events in the development of autoimmune diseases and malignancies has been investigated widely. However, the importance of apoptosis in the haemostasis of cord blood immune cells is yet to be elucidated.

Whereas adult blood T cells and neutrophils were shown to undergo different rates of apoptosis when compared to cord blood, the sensitivity of cord blood B cells to this process has not been studied. In vitro experiments demonstrate that B cells from umbilical cord are functionally distinct from adult B cells. Cord blood B cells produce small amounts of IgM and almost no IgG or IgA upon stimulation with pokeweed mitogen [1–3]. Additionally, IgG and IgA responses to viral and bacterial infections are relatively low, especially in the first months of life [4].

These immunological differences between adult and neonate undoubtedly affect the neonate's susceptibility to infection. Despite the protective effect of maternal transplacental IgG antibodies, the neonate is at risk for serious bacterial infections (SBI). The explanation for this well-known phenomenon is yet to be delineated precisely.

Immature B cell function may contribute partially to this vulnerability; moreover, enhanced apoptosis may also play a part in the increased incidence of SBI among neonates.

Finally, in umbilical cord blood 60–80% of B cells express CD5 which decreases with age to 5–30% in adulthood [5].

CD5 is responsible for the secretion of polyreactive antibodies, mainly IgM, that recognize a variety of self-antigens and foreign antigens [6]. CD5⁺ B cells are composed of two populations: CD5⁺ B cells that arise during ontogeny and CD5⁺ B cells which has been induced by various stimuli. Whereas the aforementioned B cell populations co-exist in human beings, in adult blood there is a higher proportion of induced CD5⁺ B cells [5].

Gagro et al. have demonstrated that although neonatal B cells display a greater diminution in CD5 expression than adult CD5⁺ B cells following CD40 signals, the two populations responded similarly to T dependent and independent antigens [7]. The survival of B cells and their rescue from apoptosis is related to various cytokines and factors. The cytokine interleukin (IL)-4 has been shown to prevent cell death by neglect (growth factor withdrawal) of B lymphocytes through the activation of Stat6 and subsequent transcription of Bcl-Xl [8].

Another important cell survival and maturation factor for B cells is B cell-activating factor from the tumour necrosis factor (TNF) family (BAFF), which is expressed in monocytes, macrophages and dendritic cells [9–11]. BAFF exerts its effect by binding to several receptors: transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), BAFF-R and B cell maturation Ag (BCMA) [12,13]. These receptors appear at various times during B cell differentiation; BCMA is expressed on transitional type 1 cells and plasma cells, whereas TACI and BAFF-R are expressed on transitional type 2/3 and mature B cells [14,15]. BAFF binds specifically to B cells and promotes their proliferation in the presence of anti-µ, indicating that BAFF functions as co-stimulator of B cell proliferation [9]. It was found that there is a constant requirement for BAFF-mediated signalling for both B cell maturation and survival of mature B cells [16].

In the present study, we sought to determine whether the rate of spontaneous apoptosis and cell rescue differs between B cells from cord blood and adult peripheral blood lymphocytes. To address these questions, we compared the role of CD5, IL-4, Bcl-2 and BAFF in the spontaneous apoptosis of B cells from umbilical cord blood and adult B cells.

Materials and methods

Cells

Human umbilical cord blood was obtained from the placentas of normal full-term deliveries after the placentas were delivered and separated from the infants. The parturient women, ages 22–40 years (mean 29·1 ± 5), had no acute or underlying chronic disease. Peripheral blood was obtained from 30 healthy adult hospital-worker volunteers (15 male; 15 female) whose ages ranged from 25 to 45 years (mean 33·3 ± 7).

The study was approved by the Hospital's Ethics Committee and informed consent was obtained from the participants prior to their inclusion in the study. All samples were collected in heparinized flasks.

Preparation of human mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient separation. Following isolation, they were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine, 100 µ/ml penicillin, 0·1 mg/ml streptomycin and recombinant human IL-2 at a concentration of 5 µ/ml in 24-well plates (Corning Glass Works, Corning, NY, USA) to reach a final concentration of 1 × 10⁶ cells/well.

B cell separation

PBMCs were isolated by Ficoll-Hypaque density gradient separation. B lymphocytes were positively selected with magnetic microbeads conjugated to anti-human CD22 [auto-magnetic affinity cell sorting (MACS) system, Miltenyi Biotec, Bergisch Gladbach, Germany] according to the manufacturer's instructions. Following isolation, CD22⁺ cells, comprising 95–98% of the resulting population, were cultured in RPMI-1640 supplemented with 10% FCS, l-glutamine, penicillin and streptomycin in 96-well plates (Corning Glass Works, Corning, NY, USA) to reach a final concentration of 1 × 10⁵ cells/well.

Assessment of apoptotic cell death

CD19⁺ B cell populations from cord blood and healthy donors were assessed for their sensitivity to spontaneous apoptosis. The sensitivity of B lymphocytes to be rescued by rIL-4 10 u/ml (Genzyme Corporation, Cambridge, MA, USA) during 24 h with and without the addition of BAFF (100 ng/ml) was also compared in both groups.

Flow cytometry

Annexin-V binding to B (CD19) cells was assayed by direct (one-step) triple immunostaining using FITC-conjugated monoclonal antibody (MoAb) to annexin V (annexin V-kit MedSystems Diagnostics GmbH, A-1030 Vienna, Austria), phycoerythrin (PE)-conjugated MoAb to CD19 (Genzyme Corporation) and allophycocyanin (APC)-conjugated MoAb to CD5 (DakoCytomation, Glostrup, Denmark).

Flow cytometry was carried out with a fluorescence activated cell sorter (FACS) operating with Cellquest software (Beckman Coulter). The total population of viable cells was gated according to their typical forward- and right-angle light scatter. The fluorescence of cells treated with fluorescent isotype MoAb was evaluated in each experiment to determine the level of background fluorescence of negative cells. The percentage of stained cells for annexin V and CD19 or CD19⁺ CD5⁺ was determined, following counting of a total of 50 000 cells, taken from independent replicates and taking only positive cells into account.

4′ 6-Diamidino-2-phenylindole, dihydrochloride (DAPI) staining

In order to confirm morphologically the finding of enhanced B cell apoptosis, DAPI staining was performed. Briefly, cultured B cells were washed with 1 × phosphate-buffered saline (PBS), fixed with 70% ethanol for 20 min at room temperature and washed again with 1 × PBS. Cells were then treated with DAPI (1 µg/ml; Sigma, Rechovot, Israel) at a 1 : 1000 dilution, incubated for 10 min and washed again with 1 × PBS for 5 min. Stained nuclei were visualized under a fluorescence microscope. Apoptotic cells were defined morphologically by cytoplasmic and nuclear shrinkage and chromatin condensation or fragmentation. The percentage of apoptotic cells was calculated as a proportion of 250 total cells visualized in 10 different fields of each slide/experiment.

Detection of Bcl-2 expression

Whole blood samples were stained with PE-conjugated MoAbs against surface marker CD19 (Genzyme Corporation) for 20 min. Afterwards, cells were fixed with fixation medium and permeabilized with permeabilization solution (Caltag Laboratory, Burlingame, CA, USA). Cells were then incubated with MoAbs against Bcl-2-FITC (Caltag Laboratory) for 20 min at 4°C. In the final step, cells were washed and analysed on FACS operating with Cellquest software (Beckman Coulter) for mean fluorescence intensity (MFI).

Detection of soluble BAFF protein

Recombinant soluble human BAFF, anti-BAFF MoAb and biotin-labelled anti-BAFF MoAb were provided courtesy of Biogen (Biogen Inc., Cambridge, MA, USA). Flat-bottomed enzyme-linked immunosorbent assay (ELISA) plates (Costar 9018) were coated with 50 µl of anti-BAFF moAb (clone A11.C3·4: batch no. 6423–55) at a concentration of 10 µg/ml in 50 mM bicarbonate buffer overnight at 4°C. After removal of the coating antibody, plates were washed thrice with washing buffer (0·2% Tween-20 in PBS, pH 7·2) and blocked with 100 µl of blocking buffer [5% bovine serum albumin (BSA) in PBS, pH 7·2] for 30 min at room temperature. Tested samples were diluted 1 : 2 in dilution buffer (1% BSA in PBS, pH 7·2), with 100 µl added to each well and incubated at room temperature for 1 h. After three washes, biotin-labelled anti-BAFF MoAb at a concentration of 250 ng/ml was added to each well and incubated at room temperature for 1 h. Horseradish peroxidase (HRP)-conjugated streptavidin was added after washing the plates three times at a concentration of 0·2 µg/ml and incubated for 1 h. Following the addition of HRP substrate, the colour reaction was stopped (by a stopping solution) and read at 450 nm. A standard curve was established using recombinant soluble human BAFF (batch no. 6001–133, concentration 3·04 mg/ml). BAFF levels more than 2 s.d. above the mean were considered ‘elevated’.

BAFF-receptor mRNA expression in B cells by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR)

Analysis

MasterPure™ (Epicentre, Madison, WI, USA) was used to extract RNA from B lymphocytes. cDNA was generated from 1 µg of total RNA using RT [Reverse-iT™ (AMV Rtase + MMuLV Rtase) ABgene, Surrey, UK] and random decamers (ABgene). RT products were subjected to PCR amplification with Ready Mix PCR Master Mix (ABgene) by using the following primers: BAFF-R sense primer (CTG GTC CTG GTG GGT CTG) and anti-sense primer (TCT TGG TGG TCA CCA GTT CA). The amplification conditions included denaturation at 94°C for 50 s, annealing at 57°C for 50 s and extension at 72°C for 50 s for 35 cycles. Similar conditions were used to amplify the human housekeeping gene β-actin by using the sense primer (CTC CTT AAT GTC ACG CAC GAT TTC). All primers were obtained from Proligo (Boulder, CO, USA). The cDNA products were separated on 2% agarose gel containing ethidium bromide. The amplified fragments of β-actin and BAFF-R were 530 base pairs (bp) and 256 bp, respectively. The β-actin cDNA product was used as a measure to assess whether equivalent levels of total RNA had been subjected to RT–PCR. At termination, the bands of the β-actin cDNA and those of the BAFF-R were assessed and compared.

Statistical analysis

The Kolmogorov–Smirnov test was applied in order to evaluate the normality of the distributions. Comparison of the apoptotic rates between the three matched groups with different culturing conditions in the cord B cells and in the adult B cells was performed using the analysis of variance (anova) test for repetitive measurements. When the anova P-value was significant, differences between each two groups were analysed using the paired Student's t-test corrected for the number of comparisons.

Comparison of apoptotic rates between cord B cells and adult B cells for each culturing condition was performed using Student's t-test for independent groups. Comparison of the Bcl-2 levels and BAFF serum levels between cord and adult B cells was performed using the unpaired Student's t-test. Two-tailed P-values of 0·05 or less were considered to be statistically significant.

Results

B cell apoptosis

Spontaneous apoptosis of cultured B cells, as detected by annexin V binding, was significantly higher in cord blood than in healthy adult individuals (77·5; 95% confidence interval, 73·5–81·5 versus 59·2; 95% confidence interval, 54–64, respectively, P < 0·0001).This observation was confirmed further by DAPI staining (18% versus 5%, P < 0·001) (Fig. 1).

Fig 1.

Comparison of spontaneous apoptosis (24 h) between isolated B lymphocytes from cord blood (n = 31) and peripheral adults blood (n = 17). The percentage of annexin V-staining B cells is significantly higher in cord blood (P < 0·0001). (a). A representative image of 4′ 6-diamidino-2-phenylindole, dihydrochloride (DAPI) nuclear staining of isolated B cells from peripheral adult control is shown in (b) (× 20 magnification; blue). DAPI nuclear staining of isolated B cells from cord blood is shown in (c) (× 20 magnification; blue). The arrows demonstrate apoptotic cells with condensed and fragmented nuclei.

Additionally, the apoptosis rate of CD19⁺ CD5⁺ cells was lower than CD19⁺ CD5⁻ in cord blood (25·1 ± 9·3% versus 58·5 ± 12·5%, respectively, P < 0·0001) as well as in the control group (15 ± 5·5% versus 22·7 ± 9·3%, respectively, P = 0·01) (Fig. 2). Interestingly, CD19⁺ CD5⁺ from cord blood underwent an increased rate of apoptosis in comparison to CD19⁺ CD5⁺ from the control group (25·1 ± 9·3% versus 15 ± 5·5%, respectively, P = 0·0013).

Fig 2.

Expression of annexin V on CD19⁺ CD5⁺ is significantly reduced compared with CD19⁺ CD5⁻ in peripheral blood from adult control (n = 17) and in cord blood (n = 17). Additionally, in these same subjects, expression of annexin V on CD19⁺ CD5⁺ and CD19⁺ CD5⁻ is significantly increased in cord blood compared with peripheral blood from adult control, respectively (immunostaining was performed on peripheral blood mononuclear cells).

Finally, CD19⁺ CD5⁻ from cord blood also underwent an increased rate of apoptosis in comparison to CD19⁺ CD5⁻ from the control group (22·7 ± 9·3% versus 58·5 ± 12·5%, respectively, P < 0·0001) (Fig. 2).

Intracellular Bcl-2 in B lymphocytes

Aiming to explain the increased rate of spontaneous apoptosis in cord blood B cells, we measured the expression of intracellular Bcl-2 in B lymphocytes. The MFI of intracellular Bcl-2 in B lymphocytes was higher in peripheral B blood than in cord blood (2·85 ± 1·3 versus 1·6 ± 0·9, respectively, P = 0·05) (Fig. 3).

Fig 3.

Representative plot results of intracellular expression of Bcl-2 in freshly adult B cells (n = 13) (a) and cord blood B cells (n = 9) (b).

Rescue of B cell apoptosis by IL-4

Adding soluble IL-4 10 u/ml to cultured cord or adult B cells for 24 h decreased the spontaneous apoptosis rate in both groups, but remained significantly higher in cord blood B cells (54·3; 95% confidence interval, 48·3–60·3 versus 39·3; 95% confidence interval, 34·2–44, P < 0·0001) (Fig. 4). Interestingly, the proportion (extent) of rescued B cell apoptosis among cord blood was similar to the control group (30·2; 95% confidence interval, 24·5–35·9 versus 32·0; 95% confidence interval, 27·4–36·6, respectively, P = n.s.) (Fig. 5).

Fig 4.

Spontaneous apoptosis of isolated B lymphocytes cultured with either interleukin (IL)-4 (10 u/ml) or IL-4 and B cell-activating factor from the tumour necrosis factor family (BAFF) (100 ng/ml). The percentage of annexin V staining B lymphocytes isolated from peripheral blood (n = 17) (a) or cord blood (n = 17) (b) incubated for 24 h in the presence or absence of either IL-4 or IL-4 and BAFF. The extent of B cell apoptosis was lower significantly in both groups after adding IL-4 to the cell cultures.

Fig 5.

The percentages of rescued B cells isolated from peripheral blood (n = 17) (a) and cord blood (n = 17) (b) cultured with either interleukin (IL-4) or IL-4 and B cell-activating factor from the tumour necrosis factor family (BAFF). The extent of cell rescue after incubation with IL-4 or IL-4 and BAFF was similar in both groups.

Rescue of B cell apoptosis by BAFF and IL-4

The addition of soluble recombinant BAFF (100 ng/ml) to cultured cord or adults B cells for 24 h without IL-4 10 u/ml did not provide any survival signal and spontaneous apoptosis remained unchanged.

When cord B cells and adults B cells were incubated with soluble BAFF (100 ng/ml) in addition to IL-4, we found that BAFF enhanced further B cell survival over IL-4 (Fig. 4). The rate of spontaneous apoptosis of cord B was still higher than the control group (49·0; 95% confidence interval, 43·7–54·2 versus 34·6; 95% confidence interval, 30·3–39, respectively, P = 0·00025). As with IL-4, the extent of decreased (rescued) B cell apoptosis among cord blood was similar to the adults control (10·1; 95% confidence interval, 5·5–14·6 versus 12·1; 95% confidence interval, 7·1–17, P = n.s.) (Fig. 5). Representative dot plots results of spontaneous apoptosis of isolated B cells from cord and adult blood in the different conditions (Fig. 6). In order to investigate further the role of BAFF in B cells apoptosis, we measured the expression of BAFF receptor mRNA in B lymphocytes and the levels of serum BAFF.

Fig 6.

Representative dot plots results of isolated cultured B cell apoptosis in different conditions. Spontaneous apoptosis of isolated cord blood B cells after 24 h in culture (a–c): (a) without interleukin (IL)-4 or B cell-activating factor from the tumour necrosis factor family (BAFF) (77%), (b) with IL-4 (53%) and (c) with IL-4 and BAFF (49·5%). Spontaneous apoptosis of isolated adult B cells (d–f): (d) without IL-4 or BAFF (67%), (e) with IL-4 (50·2%) and (f) with IL-4 and BAFF (44·2%).

Expression of BAFF receptor mRNA

The expression of BAFF receptor mRNA in B lymphocytes from cord blood was found to be similar to the expression in B lymphocytes from adult blood (Fig. 7).

Fig 7.

Expression of B cell-activating factor from the tumour necrosis factor family (BAFF) receptor mRNA in isolated B cells from adult (n = 3) and cord blood (n = 4).

Serum BAFF levels

However, when serum BAFF levels were compared between adult and cord blood, serum BAFF was found significantly lower in cord blood (0·68 ± 0·13 ng/ml versus 1·83 ± 0·54 ng/ml, P = 0·01) (Fig. 8).

Fig 8.

Serum B cell-activating factor from the tumour necrosis factor family (BAFF) levels in adult (n = 17) and cord blood (n = 31). Serum BAFF is significantly lower in cord blood (0·68 ± 0·13 ng/ml versus 1·83 ± 0·54 ng/ml, P = 0·01).

Discussion

The size and composition of B lymphocytes population is maintained by controlling cell growth and death. Apoptosis, an endogenous cell suicide mechanism which requires active participation of the dying cell, is the most common form of physiological cell death.

This process is important for the maintenance of B cells tolerance in the germinal centres, elimination of irrelevant B cells that may be recruited in a bystander fashion and counterselection of the somatically mutated B cell clones with low Ag-binding in the germinal centres [17]. Our present data demonstrate that cord B cells undergo increased spontaneous apoptosis compared to peripheral adults B cells. Aggarwal et al. [18] showed that there was increased T cell death in cord blood when compared to adult peripheral blood and this finding was attributed to either a low Bcl-2 expression or a low bcl-2/bax ratio. Similarly, we also found lower expression of Bcl-2 in cord B cells compared to peripheral adult B cells, which may explain the higher susceptibility of these cells to undergo apoptosis. Bcl-2 family proteins play a pivotal role in modulating membrane integrity and the release of apoptogenic factors from mitochondria. Bcl-2 localizes predominantly in the mitochondria and inhibits apoptosis. Bax, a pro-apoptotic protein, resides in the cytoplasm and stimulates cell death after translocation to the mitochondria. It appears that the equilibrium between anti-apoptotic and pro-apoptotic protein expression determines the vulnerability of the cell to apoptogenic stimuli [19].

Interestingly, most of the cord B cells possess CD5, which is known to protect B cells from activation-induced cell death and also maintain tolerance in anergic B cells in vivo [20]. It was demonstrated that CD5 provide B cells with survival signals through the down-regulation of BCR-mediated early events and enhanced IL-10 production.

IL-10 then induces Bcl-2, thereby providing a survival factor to B cells [21]. Indeed, our study demonstrates that CD5⁺ B cells in cord blood and adults are also protected from spontaneous apoptosis when compared to CD5⁻ B cells. This protection can be explained partially by Gary-Gouy et al.'s [21] finding that spontaneous IL-10 secretion by fresh CD5⁺ B cells was found to be higher than that of their CD5⁻ counterparts.

However, cord blood CD5⁺ B cells undergo increased apoptosis in comparison to adult CD5⁺ B cells. The explanation to this finding is speculative: preliminary data suggest that there may be different rates of spontaneous apoptosis between the induced and the nascent CD5-positive B cells. Given this assumption, we could attribute the decreased rate of adult CD5⁺ B cell apoptosis to be related to the higher proportion of induced CD5⁺ B cells. When the sensitivity of CD5⁻ B cells to undergo spontaneous apoptosis was also analysed, we found adult CD5⁻ B cells to be of lower sensitivity to undergo apoptosis than cord blood CD5⁻ B cells. Future studies are needed to clarify the observed differences in apoptosis between the various B cell subtypes.

Our study also sought to explore the influence of IL-4 and BAFF on B cell rescue. We found that the IL-4 and BAFF effect on cord and adult B cell rescue was similar. It is well established that BAFF attenuates the apoptosis of B cells by changing the ratios between Bcl-2 family proteins which favour cell survival. Whereas the levels of pro-apototic bak are reduced, the levels of the pro-survival counterparts, Bcl-2 and Bcl-xL, are increased. The central role of BAFF has been demonstrated in studies involving malignant B cells.

There is an increased endogenous production of BAFF in malignant B cell chronic lymphocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma B cells, whereas the rates of apoptosis were markedly decreased [22–24]. Furthermore, Do et al. [25] found that BAFF activated the nuclear factor (NF)-B transcription factors RelB and p50 in both resting and CD40L-activated B cells, suggesting that they may mediate the BAFF signals for B cell survival.

Thus, our finding that serum from cord blood contains lower levels of circulating BAFF can partially explain the lower levels of Bcl-2 and the increased susceptibility of cord blood B cells to apoptosis. Our finding of the similar expression of mRNA BAFF in both cord and adult B cells does not exclude the possibility that the expression of BAFF receptor on cord blood B cells is functionally altered. Lower serum BAFF levels in cord blood is probably related to already reported functional immaturity of cord blood macrophages (the main source of BAFF).

The mechanisms by which IL-4 rescues B cells from apoptosis have been reported in many studies. In culture, IL-4 has been shown to prevent cell death by neglect of resting B lymphocytes after growth factor withdrawal [26]. IL-4 induces production of Stat6 (transcription factor) that directly activates a Bcl-xL regulatory element, which is an important anti-apoptotic agent [27]. IL-4 can also prevent B cell induced apoptosis by Ig cross-linking that render activated B cells insensitive to Fas ligation [28,29].

Our findings are consistent with other studies that found impaired apoptosis in neonatal cells, which may contribute to prolonged inflammation in infants after oxidative stress or infection. It was found that cord blood T cells spontaneously apoptose more readily than peripheral blood T cells ex vivo [30,31]. Natural killer cells from cord blood also apoptose at higher rates than peripheral adults blood [32]. Interestingly, neonatal neutrophils which belong to the innate immune system have a decreased rate of spontaneous apoptosis compared to adult neutrophils [33,34].

In conclusion, the increased susceptibility of cord B cells to undergo apoptosis might result from the lower levels of intracellular Bcl-2 and lower levels of serum BAFF.

Increased rates of B lymphocyte apoptosis will decrease the capacity of B cells to process and present antigen to T helper cells, which is a fundamental mechanism ensuring the targeting of helper signals to the appropriate B cell [35,36]. The increased rates of B cell apoptosis observed in cord blood may render the neonate more vulnerable to infection, thereby increasing host susceptibility to enhanced morbidity and mortality from various infectious diseases.