Further differentiation of murine double-positive thymocytes is inhibited in adenosine deaminase-deficient murine fetal thymic organ culture

Murine fetal thymic organ culture (FTOC) was used to investigate the mechanism by which a lack of adenosine deaminase (ADA) leads to a failure of T cell production in the thymus. We previously showed that T cell development was inhibited beginning at the CD4(-)CD8(-)CD25(+)CD44(low) stage in ADA-deficient FTOC initiated at day 15 of gestation when essentially all thymocytes are CD4(-)CD8(-). In the present study, we asked whether thymocytes at later stages of differentiation would also be sensitive to ADA inhibition by initiating FTOC when substantial numbers of CD4(+)CD8(+) thymocytes were already present. dATP was highly elevated in ADA-deficient cultures, and the recovery of alphabeta TCR(+) thymocytes was inhibited by 94%, indicating that the later stages of thymocyte differentiation are also dependent upon ADA. ADA-deficient cultures were partially rescued by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or by the use of apoptotic protease-activating factor-1-deficient mice. Rescue was even more dramatic, with 60- to >200-fold increases in the numbers of CD4(+)CD8(+) cells, when FTOC were performed with an inhibitor of adenosine kinase, the major thymic deoxyadenosine phosphorylating enzyme, or with bcl-2 transgenic mice. dATP levels were normalized by treatment with either carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone or an adenosine kinase inhibitor, but not in cultures with fetal thymuses from bcl-2 transgenic mice. These data suggest that ADA deficiency leads to the induction of mitochondria-dependent apoptosis as a consequence of the accumulation of dATP derived from thymocytes failing the positive/negative selection checkpoint.     

Enhanced differentiation of splenic plasma cells but diminished long-lived high-affinity bone marrow plasma cells in aged mice

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.     

Function of murine adenosine deaminase in the gastrointestinal tract

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency characterized by severe T and B cell lymphopenia. ADA-deficient humans also display defective development of gut-associated lymphoid tissues (GALT). They lack lymphoid cells, and the Peyer's patches are without germinal centers. In mice, ADA-deficient fetuses die perinatally due to liver damage, but they also exhibit pathology in the thymus, spleen, and the small intestine. The GI phenotype associated with ADA-deficient humans prompted us to examine the effect of ADA-deficiency on mouse small intestine tissue. The work presented here focuses on understanding the physiological role of ADA in the GI tract, using ADA-deficient mice rescued from perinatal lethality by restoring Ada expression to trophoblast cells. Histologically and immunologically, the GALT was compromised at all sites in ADA-/- mice, with the most dramatic changes seen in the Peyer's patches. Profound disturbances in purine metabolism were detected in all the gastrointestinal tissues. In particular, adenosine and deoxyadenosine, the ADA substrates, increased markedly while the product inosine decreased. The activity of S-adenosylhomocysteine hydrolase decreased throughout the GI tract, indicating a possible disruption of cellular transmethylation and activation of apoptotic pathways. There were also disturbances in the purine metabolic pathway with a decrease in the production of downstream nucleosides hypoxanthine and xanthine.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

CD19 function in early and late B cell development: I. Maintenance of follicular and marginal zone B cells requires CD19-dependent survival signals

Loss of membrane-bound Ig results in the rapid onset of apoptosis in recirculating B cells. This observation implies that a competent B cell receptor (BCR) is not only required for Ag-dependent differentiation, but also for continued survival in the peripheral immune system. Expression of the B cell coreceptor, CD19, is likewise essential for key B cell differentiative events including the formation of B-1, germinal center, and marginal zone (MZ) B cells. In this study, we report that CD19 also exerts a role before Ag encounter by promoting the survival of naive recirculating B cells. This aspect of CD19 signaling was first suggested by the analysis of mixed bone marrow chimeras, wherein CD19-/- B cells fail to effectively compete with wild-type B cells to reconstitute the peripheral B cell compartment. Consistent with this observation, Bromodeoxyuridine- and CFSE-labeling studies reveal a shorter in vivo life span for CD19-/- B cells vs their wild-type counterparts. Moreover, we find that CD19 is necessary for propagation of BCR-induced survival signals and thus may contribute to homeostatic mechanisms of tonic signaling. To determine whether provision of a constitutive survival signal could compensate for the loss of CD19 in vivo, Bcl-2-transgenic mice were bred onto the CD19-/- background. Here, we observe an increase in follicular B cell numbers and selective recovery of the MZ B cell compartment. Together these findings suggest that maintenance of the follicular and MZ B cell compartments require CD19-dependent survival signals.     

Abnormal immune function of hemopoietic cells from alymphoplasia (aly) mice, a natural strain with mutant NF-kappa B-inducing kinase

Alymphoplasia (aly) mice, a natural strain with a mutant NF-kappa B-inducing kinase (NIK) gene, manifest a unique phenotype; they lack lymph nodes and Peyer's patches, have a disturbed spleen architecture, and exhibit defects in both Ab and cellular immune responses. Although a stromal defect caused by impaired lymphotoxin-beta receptor signaling accounts for their abnormal lymphoid organogenesis, the exact mechanisms underlying the development of immunodeficiency in aly mice are poorly understood. We therefore investigated the contribution of hemopoietic cells with the aly NIK mutation to the development of immunodeficiency. Transfer of aly/aly bone marrow cells into aly/+ mice resulted in poorly developed B cell follicles and lack of support for the development of germinal centers and isotype switching, indicating that the hemopoietic cells of aly mice contain an autonomous defect. However, follicular dendritic cell clusters were maintained in the spleens of these bone marrow chimeras, suggesting that the lack of follicular dendritic cell clusters in aly mice is probably due to the stromal defect. The aly mice lacked marginal zone B cells in their spleens, and aly/aly B cells showed an impaired proliferative response after in vitro stimulation. IL-2 production by activated T cells was also impaired. By contrast, the dendritic cells of aly mice exhibited grossly normal development and function. Supporting the concept of an autonomous cell defect, Rel protein expression was altered in aly/aly spleens. Thus, the aly NIK mutation affects hemopoietic cell function in an intrinsic fashion and, together with the stromal defect, may contribute to the development of immunodeficiency in aly mice.     

Abrogation of hybrid resistance to bone marrow engraftment by graft-vs-host-induced immune deficiency

Lethally irradiated F1 mice, heterozygous at the hematopoietic histocompatibility locus Hh-1, which is linked with H-2Db, reject bone marrow grafts from H-2b parents. This hybrid resistance (HR) is reduced by prior injection of H-2b parental spleen cells. Because injection of parental spleen cells produces a profound suppression of F1 immune functions, we investigated whether parental-induced abrogation of HR was due to graft-vs-host-induced immune deficiency (GVHID). HR was assessed by quantifying engraftment of H-2b bone marrow in F1 mice with the use of splenic [125I]IUdR uptake; GVHID, by the ability of F1 spleen cells to generate cytotoxic T lymphocytes (CTL) in vitro. We observed a correlation in the time course and spleen cell dose dependence between loss of HR and GVHID. Both GVHID and loss of HR were dependent on injection of parental T cells; nude or T-depleted spleen cells were ineffective. The injection of B10 recombinant congenic spleens into (B10 X B10.A)F1 mice, before grafting with B10 marrow, demonstrated that only those disparities in major histocompatibility antigens that generated GVH would result in loss of HR. Thus, spleens from (B10 X B10.A(2R]F1 mice (Class I disparity only) did not induce GVHID or affect HR, whereas (B10 X B10.A(5R))F1 spleens (Class I and II disparity) abrogated CTL generation and HR completely. GVHID produced by a class II only disparity, as in (B10 X B10.A(5R))F1 spleens injected into (B6bm12 X B10.A(5R))F1 mice, was also sufficient to markedly reduce HR to B10 bone marrow. This evidence that GVHID can modulate hematopoietic graft rejection may be relevant to the mechanisms of natural resistance to marrow grafts in man.     

In vitro and in vivo activation induces BAFF and APRIL expression in B cells

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88-dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up-regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmembrane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB x NZW)F(1) mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB x NZW)F(1) mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.     

Lymphocyte accumulation in the spleen of retinoic acid receptor-related orphan receptor gamma-deficient mice

The hormone nuclear receptor retinoic acid receptor-related orphan receptor gamma (RORgamma) plays important roles in thymocyte development and lymphoid organogenesis. RORgamma and its thymus-specific isoform RORgammat are expressed in the thymus, but not in the spleen and bone marrow (BM). However, RORgamma(-/-) mice have 2- to 3-fold more splenocytes than wild-type controls due to an accumulation of conventional resting B lymphocytes. The increase in B lymphocytes in RORgamma(-/-) mice is caused neither by abnormal B cell development in the BM nor by an obvious defect in the peripheral T cell compartment. Furthermore, analyses of BM chimeras using either RORgamma(-/-) or recombinase-activating gene-2(-/-) mice as recipients and wild-type or RORgamma(-/-) mice as donors, respectively, demonstrate that the splenic microenvironment of RORgamma(-/-) mice is defective, since wild-type T and B lymphocytes accumulated in these chimeric mice. In addition, T lymphocyte homeostasis was altered due to a lowered thymic output in RORgamma(-/-) mice. Collectively, these results suggest that RORgamma regulates lymphocyte homeostasis at multiple levels.     

Characterization of normal and mutant adenosine deaminase messenger RNAs by translation and hybridization to a cDNA probe

Summary Using both in vitro translation and hybridization to an adenosine deaminase (ADA) cDNA probe, ADA mRNA has been characterized in B lymphoblast lines established from seven ADA-deficient children, two parents of an ADA-deficient child, and three normal people. All ADA-deficient lines except GM-2825A, including those with less than 1% of normal catalytic activity, had normal or greater amounts of hybridizable, 1.6 kilobase in size, ADA mRNA. Immunoreactive ADA protein of normal size was produced by in vitro translation of the mRNAs. Deficiency of ADA activity in these lines appears secondary to synthesis of structurally altered proteins rather than to a quantitative deficiency in ADA mRNA. The GM-2825A line contains electrophoretically abnormal species of RNA which hybridize to the cDNA probe. Deficiency of ADA activity in this line appears at least in part secondary to a structural defect in the ADA mRNA or its precursors.     

Rapid induction of splenic and peritoneal B-1a cells in adult mice by thymus-independent type-2 antigen

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

Mice with a targeted mutation in lymphotoxin-alpha exhibit enhanced tumor growth and metastasis: impaired NK cell development and recruitment.

Mice deficient in lymphotoxin (LT)-alpha lack peripheral lymph nodes and Peyer's patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LTalpha is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LTalpha for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LTalpha-deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LTalpha-/- mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LTalpha-/- mice. Although LTalpha-/- mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LTalpha-/- bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LTalpha signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.     

Impaired germinal center reaction in mice with short telomeres

Reduction of germinal center reactivity is a landmark of immunosenescence and contributes to immunological dysfunction in the elderly. Germinal centers (GC) are characterized by extensive clonal expansion and selection of B lymphocytes to generate the pool of memory B cells. Telomere maintenance by telomerase has been proposed to allow the extensive proliferation undergone by B lymphocytes in the GC during the immune response. We show here that late generation mTR(-/-) mice, which lack the mouse telomerase RNA (mTR) and have short telomeres, present a dramatic reduction in GC number following antigen immunization. Upon immunization with an antigen, wild-type splenocyte telomeres are elongated and this is accompanied by a high expression of the telomerase catalytic subunit in the spleen GC. In contrast, telomerase-deficient mTR(-/-) splenocytes show telomere shortening after immunization, presumably due to cell proliferation in the absence of telomerase. All together, these results demonstrate the importance of telomere maintenance for antibody-mediated immune responses and support the notion that telomere elongation detected in wild-type spleens following immunization is mediated by telomerase.     

Correction or transfer of immunodeficiency due to TNF-LT alpha deletion by bone marrow transplantation.

BACKGROUND: Mice with inactivated tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) genes have profound abnormalities of the immune system including lymphocytosis, lack of lymph nodes, undifferentiated spleen, hypoimmunoglobulinaemia, and defective Ig class switch. Here, we asked whether this phenotype is due to incompetent lymphohemopoietic progenitors or to a defective environment. MATERIALS AND METHODS: Lethally irradiated TNF-LT alpha-deficient and wild-type mice received bone marrow cells from either TNF-LT alpha-deficient or wild-type mice. The reconstitution and transfer of the phenotype was followed by morphological and functional analyses. RESULTS: Bone marrow cells from wild-type mice restored the synthesis of TNF and LT alpha, corrected the splenic microarchitecture, normalized the lymphocyte counts in the circulation, and repopulated the lamina propria with IgA-producing plasma cells of TNF-LT alpha-deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell dependent antigen was corrected, while no lymph nodes were formed. Conversely, the TNF-LT alpha phenotype could be transferred to wild-type mice by bone marrow transplantation after lethal irradiation. CONCLUSIONS: These data demonstrate that most TNF- and LT alpha-producing cells are bone marrow derived and radiosensitive, and that the immunodeficiency due to TNF-LT alpha deletion can be corrected to a large extent by normal bone marrow cell transplantation. The genotype of the donor bone marrow cells determines the functional and structural phenotype of the TNF-LT alpha-deficient adult murine host, with the exception of lymph node formation. These findings may have therapeutic implications for the restoration of genetically defined immunodeficiencies in humans.     

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.     

Genetic removal of the A2A adenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice

Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A(2A) adenosine receptor (A(2A)R) functions to limit inflammation and promote tissue protection; however, the contribution of A(2A)R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A(2A)R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A(2A)R double knockout mice. Genetic removal of the A(2A)R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A(2A)R. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-alpha and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A(2A)R double knockout mice. These findings suggest that the A(2A)R plays a protective role in the ADA-deficient model of pulmonary inflammation.