The bulk of the peripheral B-cell pool in mice is stable and not rapidly renewed from the bone marrow.

Abstract

We have reevaluated the in vivo lifespan of B lymphocytes based on incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine into the DNA of dividing cells. To exclude potential alterations in the turnover of B-lineage cells due to the bromodeoxyuridine incorporation, we performed pulse-chase experiments comparing the appearance and disappearance of bromodeoxyuridine-labeled cells over long periods of time. The data consistently show that more than two-thirds of splenic B cells in adult (>2 months old) mice have a lifetime of several weeks or months, whereas a more rapid turnover takes place in young (4 week old) mice. Thus, the peripheral B-cell pool is only slowly renewed after it has been initially built up early in life.While it is known that in the bone marrow of mice roughly 10 million B cells are produced per day (1), it is still controversial to which extent these cells renew the peripheral B cell pool (consisting of 10 times as many cells) (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). By using flow cytometry for the simultaneous detection of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and cell surface marker expression, we found (13) that, in mice fed BrdUrd for 1 week, less than 20% of splenic B cells were labeled with the drug (13), in agreement with data obtained in rats (11). Administration of BrdUrd in vivo, however, requires careful control for toxic effects on the animal's cells because of the potential mutagenicity of the drug (14), its potential interference with cellular differentiation processes, or both (15). In this context, a critical requirement is that the proliferative behavior of BrdUrd-labeled cells is not different from that of unlabeled cells. In the present experiments, therefore, we determined the turnover of B-lineage cells in a criss-cross fashion by either measuring the appearance of BrdUrdlabeled B cells after continuous BrdUrd treatment or by determining the loss of BrdUrd-labeled cells in mice whose lymphocytes had initially been labeled with BrdUrd. In addition, we compared B-cell turnover in young versus adult mice. MATERIALS AND METHODSMice. BALB/c and CB.20 mice were bred in our animal colony and were used at either 4 weeks or 2-4 months of age.Bone Marrow Reconstitution. CB.20 mice were irradiated with 600 rads (1 rad = 0.01 Gy) and reconstituted with BALB/c bone marrow on the following day. For this purpose, bone marrow cells were isolated from adult BALB/c donors by flushing the femur with RPMI 1640 medium containing 5% (vol/vol) fetal calf serum. The cells were washed in phosphate-buffered saline (PBS) and 5 x 106 cells per mouse were injected intravenously.BrdUrd Treatment. Mice were given drinking water containing BrdUrd (Sigma, B5002) at 1 mg/ml for the indicated periods of time. Drinking water containing BrdUrd was protected from light and exchanged every 3-4 days.Staining and Flow Cytometric Analysis. The staining procedure as well as the reagents used have been described (13). Briefly, 5 x 106 spleen or bone marrow cells were first stained on the surfa...

B Cells Expressing a Natural Polyreactive Autoantibody Have a Distinct Phenotype and Are Overrepresented in Immunoglobulin Heavy Chain Transgenic Mice

“…9). The percentages of BrdU labeling are consistent with those of the previously published data in normal adult mice (54,64). Therefore, the ppc1-5H/ 1 NAA B cells have a lifespan comparable to that of mature peripheral B cells.…”

“…BrdU is a thymidine analog that is incorporated into DNA when cells divide. In rapidly dividing cells such as bone marrow immature B cells, BrdU uptake is a measurement of cell proliferation; in nondividing cells such as the bulk of splenic mature B cells (64), it is a measurement of cell turnover or renewal. The turnover rate inversely correlates with the lifespan of the B cells (54, 64 -66).…”

B Cells from Mice Prematurely Expressing Human Complement Receptor Type 2 Are Unresponsive to T-Dependent Antigens

“…Of these, only 10 -20% reach the periphery, and as few as 3% contribute to the mature B cell pool (1)(2)(3)(4). This illustrates the fundamental mechanism that has evolved to regulate the generation of autoreactive B cells, namely negative selection through apoptosis.…”

Estimating average cellular turnover from 5–bromo–2'–deoxyuridine (BrdU) measurements

“…The base analogue 5-bromo-29-deoxyuridine (BrdU) can be administered via drinking water and substitutes for thymidine in newly synthesized DNA. BrdU 1 cells can be easily detected by flow cytometry, and BrdU labelling has been widely used to study lymphocyte kinetics in animals (Gratzner 1982;Penit & Vasseur 1988;Forster et al 1989;Forster & Rajewsky 1990;Rocha et al 1990;Schittek & Rajewsky 1990;Tough & Sprent 1994Dolbeare 1995;Zimmerman et al 1996;Mohri et al 1998;Bonhoeffer et al 2000;Kovacs et al 2001). Cellular turnover rates in man have also been studied by the administration of deuterated glucose ( 2 H-glucose), which leads to deuterium being incorporated into the DNA of dividing cells (Macallan et al 1998;Hellerstein 1999;Hellerstein et al 1999;McCune et al 2000;Mohri et al 2001;Ribeiro et al 2002a,b).…”