Immune deficiencies, infection, and systemic immune disorders
Effects of purine nucleoside phosphorylase deficiency on thymocyte development

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Background

Inherited or acquired defects in purine nucleoside phosphorylase (PNP) impair purine metabolism, as well as the survival and function of T lymphocytes. However, the effects of PNP deficiency on thymocyte development are not well known.

Objectives

We sought to study thymocyte development in PNP-deficient (PNP-KO) mice.

Methods

Maturation, proliferation, and apoptosis were determined in thymocytes from PNP-KO mice and hematopoietic stem cells from these mice grown ex vivo into thymocyte-like cells.

Results

Reduced percentages of CD4+CD8+ double-positive (DP) thymocytes with normal percentages of CD4−CD8+ and CD4+CD8− single-positive thymocytes were found in the thymi of PNP-KO mice. Similarly, reduced DP-like thymocytes grew ex vivo from hematopoietic stem cells of PNP-KO mice. Thymi of PNP-KO mice contained increased apoptotic DP thymocytes. Increased apoptosis of PNP-deficient DP thymocytes occurred after exposure to deoxyguanosine (dGuo), although not after Fas ligation, and could be prevented by restoring PNP activity within the cells. In DP thymocytes from PNP-KO mice, dGuo caused mitochondrial membrane potential dissipation and induced release of cytochrome c from the mitochondria followed by nuclear DNA fragmentation. Inhibition of the caspase pathway prevented dGuo-induced nuclear DNA fragmentation but not mitochondrial membrane potential dissipation, indicating that PNP deficiency induces apoptosis that is initiated in the mitochondria of DP thymocytes. 5-Bromo-2-deoxyuridine incorporation demonstrated that PNP deficiency does not interfere with DP or single-positive thymocyte proliferation.

Conclusions

PNP is important for the survival of DP thymocytes. Accumulation of dGuo in cases of PNP deficiency leads to mitochondria-initiated apoptosis of DP thymocytes, which can be prevented by restoring PNP activity in the cells.

Section snippets

Mice

Six-week-old PNP-KO (C57BL/6) mice and normal littermates maintained in a pathogen-free environment at the animal facility of the Hospital for Sick Children, Toronto, Canada, were used for all experiments. PNP deficiency was determined based on the results of Southern blot analysis and the absence of PNP enzyme activity in tail blood samples.5 The institution’s Animal Care Committee approved all procedures in accordance to the Canadian Council of Animal Care Guidelines.

PNP activity and PNP fused to TAT protein transduction domain

PNP activity was measured

Effects of PNP deficiency on thymocytes

To investigate the effects of PNP deficiency on thymocytes, we used the PNP-KO murine model. Similar to PNP-deficient patients,18 the thymi of PNP-KO mice were small (0.062 ± 0.023 g compared with 0.085 ± 0.021 g in normal littermates, P < .001, n = 50 in each group). The thymi of 6- to 8-week-old PNP-KO mice also contained only 51.4 ± 14.1 × 106 thymocytes compared with 169.9 ± 32.6 × 106 thymocytes in normal control animals (P < .001, n = 15 in each group). Compared with normal littermates,

Discussion

Studying the effect of PNP deficiency on thymocytes has been previously hindered by the limited availability of thymic samples. Here we overcame this obstacle by taking advantage of our access to thymocytes from PNP-KO mice and our ability to generate PNP-deficient thymocyte-like cells from the bone marrow of these mice. A striking finding is the effect of PNP deficiency on DP thymocytes. DP thymocytes and DP-like cells in the thymus and in ex vivo cultures of PNP-KO mice, respectively, were

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    • Cited by (0)

    Supported by grants from the March of Dimes (no. 6-FY07-326), the Canadian Centre for Primary Immunodeficiency, the Canadian Immunodeficiency Society, and the Jeffrey Modell Foundation.

    Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

    ∗

    These authors contributed equally to this work.

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    Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.