Bone marrow microenvironmental changes in aged mice compromise V(D)J recombinase activity and B cell generation

doi:10.1016/j.smim.2005.05.012

Seminars in Immunology

Volume 17, Issue 5, October 2005, Pages 347-355

https://doi.org/10.1016/j.smim.2005.05.012 Get rights and content

Abstract

B cell generation and immunoglobulin (Ig) diversity in mice is compromised with aging. Our recent work sought to understand mechanism(s) that contribute to reduced B cell production in aged mice. Using in vivo labeling, we found that reduction in marrow pre-B cells reflects increased attrition during passage from the pro-B to pre-B cell pool. Analyses of reciprocal bone marrow (BM) chimeras reveal that the production rates of pre-B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes in pro-B cells that could diminish production of pre-B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro-B cells at the single cell level. The percentage of pro-B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J recombinase activity in pro-B cells and reduced numbers of pre-B cells. Reciprocal BM chimeras revealed that the aged microenvironment also determines rag2 expression and recombinase activity in pro-B cells. These observations suggest that extrinsic factors in the BM that decline with age are largely responsible for less efficient V(D)J recombination in pro-B cells and diminished progression to the pre-B cell stage. These extrinsic factors may include cytokines and chemokines derived from BM stromal cells that are essential to the development of B cell precursors. The changes during aging within the BM hematopoietic microenvironment most likely are linked to the physiology of aging bone. Bone degrades with age (osteoporosis) due to decreased formation of new bone by osteoblasts. Marrow stem cells (MSC) are considered the progenitor of both adipocytes, osteoblasts and hematopoietic stromal cells and a controlled reciprocal regulation exists of osteoblast versus adipocyte differentiation; with age adipocytes increase, and osteoblast decrease. It is possible that stromal cell generation from MSC is compromised during aging. Currently, understanding of BM microenvironmental factors that regulate rag gene expression is very limited. However, as early progenitors differentiate, it is increasing clear that a limited set of transcription factors (e.g. ikaros, PU.1, E2A, EBF, pax5) regulate B-lineage specific genes, and that expression and stability of these factors is responsive to the microenvironment. Current and future work by several groups will strive to understand mechanisms that regulate these factors and how aging impacts these regulatory circuits.

Section snippets

Humoral immunity, aging and B cell development

The renewal of the immune system in mice declines during aging: B and T cell generation are reduced [1], [2] and as a result, old mice are more susceptible to pathogens [3]. The human immune system is also compromised during aging, resulting in increased susceptibility to pathogens and reduced vaccine efficacy [2], [4]. Given the critical role of humoral immunity for both vaccines and responses to infection, we have used murine models to ask whether the generation of the primary B cell

V(D)J recombination in B cell development

The generation of new B cells is completely dependent on the assembly of Ig genes by V(D)J recombination. In each B cell progenitor, a unique combination of V_H, D_H and J_H gene segments (one of each segment) are joined together to encode the IgH variable region, and either V_κ and J_κ or V_λ and J_λ gene segment are joined to encode the IgL variable region. Each V, D and J segment is flanked by a conserved recombination signal sequence (RSS) that are the targets of the V(D)J recombinase (reviewed in

B cell development is attenuated in aged mice due to decreased pre-B cell generation

B cell generation and frequencies of B cell subsets undergo age-associated attenuation in mice: B cell generation is markedly reduced [1], [45], and fewer mature B cells are produced. However, the number of mature B cells is similar to that of young mice due to a significant increase in the half-life of mature cells [1], [4]. Frequencies and numbers of B cell progenitor subsets in the bone marrow also change with age. The most noted change is the reduced frequency and number of pre-B cells,

Microenvironment defects underlie reduced B cell development with age

The decrease in numbers of pre-B cells in aged mice could be due to either cell-intrinsic defects in the B cell precursors themselves or alterations in the developmental microenvironment. Contact-mediated signals from bone marrow stromal cells and soluble factors are required for commitment, development, proliferation and survival of developing B cell precursors (reviewed in [56], [57], [58]). Accordingly, stromal cell cultures established from aged mice are less supportive of proliferation and

Age-associated reduction in pre-B cells is correlated with a reduction in the percent of pro-B cells that express rag2

The decreased generation of pre-B cells suggested to us that the molecular defects in aged pro-B cells might include diminished V(D)J recombination that could in turn be responsible for decreased efficiency of generating functional IgH to serve as a component of the pre-BCR. When measured in the total bone marrow of aged mice, rag1 and rag2 mRNA levels decline dramatically [9], [50], [59]. However, the age-related decline in rag expression could have been due to a global loss of bone marrow

rag2 expression, V(D)J recombinase activity and the pre:pro ratio are reduced in aged mice

Using H2-SVEX transgenic mice, we also found that reduced expression of rag2 in aged mice yields a corresponding decrease in recombinase activity [60]. Cells that undergo V(D)J recombination of this transgenic recombination substrate express the GFP variant VEX [61], [62], are easily detected by flow cytometry and are readily resolved from cells expressing conventional GFP [61], [62].

We have established that the percent of VEX⁺ cells within a population reflects the level of rag2 expression [60]

The bone marrow microenvironment also controls rag2 expression and V(D)J recombinase activity in pro-B cells

To determine if these age-associated alterations are microenvironmental versus cell intrinsic, we again used adoptive transfers. We observed that young donor-derived pro-B cells displayed lower rag2 expression and recombinase activity (and a lower pre:pro ratio) following bone marrow transfer into aged as compared to young recipient mice [55]. This indicates that age-associated alterations specific to the bone marrow microenvironment are sufficient to produce these defects in B cell

Potential bone marrow microenvironment defects in aging

The bone marrow microenvironment includes factors derived from bone marrow stromal cells that are essential to the development of B cell precursors. As specific extrinsic factors that induce rag expression have yet to be identified, the age-related decrease in rag2 expression in pro-B cells might reflect attenuation of yet unknown inductive signals. The nature of these changes remains to be determined, although rapid progress is being made in understanding the control of B cell development by

Transcription factors regulate B-lineage specific genes, including rag1 and rag2

Currently, understanding of BM microenvironmental factors that regulate rag gene expression is very limited. However, as early progenitors differentiate, it is increasing clear that a limited set of transcription factors regulates B-lineage specific genes. Key transcription factors implicated in controlling early stages of lymphopoiesis include ikaros, PU.1, E2A, EBF and pax5 [94], [95], [96], [97], [98]. Of these, E2A, EBF and pax5 are required for commitment to the B lineage (reviewed in [99]

Acknowledgements

We thank Alex Sah, David Allman, Michael Cancro (UPenn) for their invaluable contribution to our collaboration, and Robert Woodland, Lyn Schmidt and Janet Stavnezer (UMMS) as well as Bonnie Blomberg and Richard Riley (U. Miami) for helpful discussions. This work was supported in part by NIH grants AI043534 and AG19042 to R.M.G., and NIDDK 5 P30 DK32520 to the UMMS Diabetes and Endocrinology Center.

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¹: Present address: Department of Immunology, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15261, USA.

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Bone marrow microenvironmental changes in aged mice compromise V(D)J recombinase activity and B cell generation

Abstract

Section snippets

Humoral immunity, aging and B cell development

V(D)J recombination in B cell development

B cell development is attenuated in aged mice due to decreased pre-B cell generation

Microenvironment defects underlie reduced B cell development with age

Age-associated reduction in pre-B cells is correlated with a reduction in the percent of pro-B cells that express rag2

rag2 expression, V(D)J recombinase activity and the pre:pro ratio are reduced in aged mice

The bone marrow microenvironment also controls rag2 expression and V(D)J recombinase activity in pro-B cells

Potential bone marrow microenvironment defects in aging

Transcription factors regulate B-lineage specific genes, including rag1 and rag2

Acknowledgements

Cell Immunol

Exp Gerontol

Immunol Today

Cell Immunol

Curr Opin Immunol

Trends Immunol

Cell

Immunity

Mol Immunol

Blood

Blood

Blood

Mol Immunol

Mol Immunol

Mol Immunol

Immunity

Immunity

Cell

Cell

Immunity

Immunity

Clin Immunol Immunopathol

Exp Gerontol

Blood

Semin Immunol

Curr Opin Immunol

Immunity

Cell

Blood

Immunity

Cancer Cell

Mech Ageing Dev

Immunity

Reprod Biomed Online

J Biol Chem

Immunity

Bone

Blood

Immunity

Immunity

Cell

Immunity

Dev Cell

B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation

J Immunol

The aging immune system: primer and prospectus

Science

Unexpected expansions of CD8-bearing cells in old mice

J Immunol

Immunosenescence and germinal center reaction

Immunol Rev

Altered VH gene segment utilization in the response to phosphorylcholine by aged mice

J Immunol

Effect of age on humoral immunity, selection of the B-cell repertoire and B-cell development

Immunol Rev

Impaired regeneration of the peripheral B cell repertoire from bone marrow following lymphopenia in old mice

Eur J Immunol