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For permissions, please email: journals. Issue Section:. Download all slides. In contrast, mice lacking TNAP generated by homologous recombination using embryonic stem ES cells have normal skeletal development. However, at approximately two weeks after birth, homozygous mutant mice develop seizures which are subsequently fatal.
Defective metabolism of pyridoxal 5'-phosphate PLP , characterized by elevated serum PLP levels, results in reduced levels of the inhibitory neurotransmitter gamma-aminobutyric acid GABA in the brain. The mutant seizure phenotype can be rescued by the administration of pyridoxal and a semi-solid diet.
Moreover, we show the consequences of PAP deficiency alone and combined with the absence of CD73 to the composition of immune cells in main lymphoid organs. To the best of our knowledge, this is the first demonstration of a role for PAP in the immune system. Two- to 4-month-old mice were used.
The mice were maintained in a specific pathogen-free stage at Central Animal Laboratory at the University of Turku, complying with international guidelines on the care and use of laboratory animals and performed in compliance with the 3Rs principle. The bound rhCD73 was eluted using 0. Enzyme specific activity for the dephosphorylation of p-nitrophenylphosphate at pH 4.
Single cell suspensions were prepared from minced pieces of peripheral lymph nodes pooled axial and inguinal nodes , thymus, and spleen by mechanical teasing through a metal meshwork and bone marrow cells by flushing the femurs. Blood was collected by cardiac puncture. Erythrocytes were removed from spleen and blood by hypotonic lysis.
Representative dot blots and histograms were made with FlowJo software. Tissue sections were also stained with hematoxylin and eosin. Measurement of hPAP activity revealed efficient hydrolysis of artificial substrate pNPP by the purified enzyme Figure 1 a and further demonstrated its ability to metabolize [ 3 H]AMP Figure 1 b , with highest catalytic activities being detected at acidic pH.
The double knockouts generated for this work did not show any obvious phenotype, when housed in specific pathogen-free conditions. No significant shifts in other purine-converting enzymes, adenosine deaminase and adenylate kinase, were detected among the studied genotypes Figure 3. The distribution of different nucleotidases in the lymphoid tissues was then evaluated in situ by a lead precipitation method.
Short-time incubation of mouse lymph node and human tonsil cryosections with different nucleotide substrates revealed the presence of high AMPase and especially ATPase activities Figure 4. The employment of TMP as a substrate and increasing the incubation time also allowed us to detect relatively moderate but clearly visible staining, which was specifically restricted to the subcapsular area of mouse lymph nodes, the epithelial layer of human tonsils, and also individual lymphoid cells scattered throughout the lymphoid tissues Figure 4.
Strikingly, the most intense TMPase staining was observed at acidic pH, suggesting the predominant contribution of PAP enzyme to the measured nucleotidase activity. Noteworthy, it has been proposed recently that selective expression of tissue-nonspecific alkaline phosphatase and another pH-dependent enzyme tartrate-resistant acid phosphatase TRAP in corresponding alkaline and acidic lacunas of bone might function as specific pH sensors directionally regulating nucleotide receptor-mediated osteoclast function and bone resorption [ 28 ].
It may be reasonably speculated that different localization of CD73 and PAP and their inverse pH dependence provide efficient means for tuned regulation of purine homeostasis in lymphoid tissues during inflammation, hypoxia, and other patho physiological states. Further studies are required to validate this hypothesis.
As there is no reliable antibody to analyze PAP expression at the protein level in mouse, we were mining the ImmGen consortium data bank that contains detailed analyses of different murine leukocyte and endothelial cell populations in respect to their gene expression profiles.
A simplified comparison to CD73 expression is shown in Table 1. The same held true when the lymphocyte populations were analyzed in blood and bone marrow of 8-month-old PAP mice data not shown.
We chose this time point as PAP knockout mice develop nonmetastatic prostate carcinoma at an old age [ 30 ]. Also salivary glands contain more immune cells in these male mice, when they are young, but the difference disappears with aging [ 31 ].
The practically nonexisting alterations of major immune cell subpopulations in absence of PAP suggest that PAP does not seem to have any marked role in the immune system or its absence is compensated by CD Likewise, the changes in CD73 knockout mice were minor Figures 5 a — 5 d.
Fewer B cells and slightly increased number of regulatory T cells were detected in peripheral lymph nodes. In single knockouts, the number of regulatory T cells was significantly decreased in thymus and in the double knockouts both in PLN and thymus Figures 5 e and 5 f.
Still the intensity of FoxP3 expression, a marker for regulatory T cells, was at the same level in both double knockouts and wild type mice Figure 5 e. Although adenosine produced by CD73 on regulatory T cells propagates generation of more regulatory T cells via adenosine receptor [ 32 ], the number of regulatory T cells is very low and the main adenosine may come from peripheral lymph node stromal cells such as from endothelial cells.
Our finding of normal levels of regulatory T cells in organs other than in thymus, which had decreased number of regulatory T cells, and in lymph nodes, which had slightly increased number of regulatory T cells, is in agreement with the observation of Ehrentraut et al. This demonstrates that expression of CD73 overall is not needed for generation of regulatory T cells as such, although it is fundamental for the immunosuppressive function of these cells [ 9 , 33 ].
As thymus is the central organ in T cell development, we also analyzed thymic morphology and T cell subtypes of CD73, PAP, and double knockouts and compared them to those of wild type mice. The double knockout mice had normal thymic morphology Figure 6 a and tissue location of different cell types was comparable between the genotypes.
Lymph nodes, spleen, and blood represent anatomically and functionally different environments for leukocytes providing unique cellular networks and extracellular milieu. Therefore, our results showing organ-specific changes in lymphocyte subpopulations of double knockouts are understandable and emphasize the fact that, for example, in lymph nodes and spleen the factors promoting activation and differentiation of regulatory T cells are at least partially different and in spleen not markedly regulated by CD73 and PAP, although both enzymes are present in these organs.
Regulatory T cells are produced in thymus and from there they disperse throughout the body to various organs in the hematolymphoid system or differentiated in the periphery.
Regulatory T cells are heterogeneous regarding their phenotype, function, and epigenetic status and also the homing-associated molecules on regulatory T cells vary [ 34 , 35 ]. This allows them to preferentially migrate to different organs such as peripheral lymph nodes, gut-associated lymphatic tissues, spleen, or nonlymphoid organs depending on the pattern of homing-receptors on their surface [ 35 ].
For example, CXCL12 highly expressed in bone marrow and splenic red pulp attracts regulatory T cells, while CCL25 recruits them to the small intestine. Thus, the regulatory T cells in different organs have phenotypically unique characteristics. Generation and maintenance of the balance between different leukocyte subpopulations is a key element for proper functioning of the immune system in health and diseases. Although adenosine is important propagating regulatory T cells, lack of CD73 has surprisingly little effect on the number of regulatory T cells.
This is the first report demonstrating the distribution of PAP in various mouse and human lymphoid tissues and its contribution, in conjunction with CD73, to the lymphoid purine homeostasis and the balance of leukocyte subpopulations in various organs. However, alone absence of PAP does not have any significant effect outside the thymus, but, combined to the deficiency with CD73, the consequences become significant.
Further work is needed to elucidate the exact mechanism of the mode of action of PAP in the immune system. The authors declare that there is no conflict of interests regarding the publication of this paper. They are grateful to Dr. Heikki Irjala for providing the samples of human tonsils and to Anne Sovikoski-Georgieva for secretarial help.
This work benefitted from data assembled by the ImmGen consortium. Yegutkin et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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