RESUMO
Lymphopenia is a characteristic of zinc deficiency, which is associated with massive loss of pre-B and pre-T cells from the primary lymphoid organs of zinc-deficient mice that have elevated serum corticosterone (CS). We examined whether this naturally elevated glucocorticoid level is associated with increased apoptotic loss of pre-T cells in the thymus of A/J and CAF1/J mice. In three experiments, partially atrophied thymuses were removed from 20 marginally zinc-deficient (ZD) young adult mice and cultured for 6 h in parallel with thymocytes prepared from 17 adequately fed mice. Thymocyte immunophenotyping combined with flow cytometric cell cycle analysis was used to identify the degree of apoptotic cell death among thymocytes of the two dietary groups, which were compared in the absence of in vivo phagocytosis. Apoptosis was enhanced 50-300% among pre-T cells (CD4+CD8+) prepared from ZD mice. This resulted in a 38% shrinkage of the thymic pre-T cell compartment, which was associated with an 80% decrease in thymic cell number. Pro-T cells (CD4-CD8-) and mature T cells (CD4+CD8-, CD4-CD8+), which express higher levels of Bcl-2 protein, survived ZD to a greater extent and formed a greater proportion of the remaining thymocyte population in ZD mice. Collectively, these data show that heightened degrees of apoptotic cell death induced in vivo by CS-disrupted thymic T cell lymphopoiesis, identifies the means of disruption of marrow B cell lymphopoiesis and explains the appearance of lymphopenia.
Assuntos
Apoptose/fisiologia , Linfopenia/etiologia , Subpopulações de Linfócitos T/fisiologia , Timo/citologia , Zinco/deficiência , Animais , Apoptose/imunologia , Peso Corporal , Citometria de Fluxo , Imunofenotipagem , Camundongos , Tamanho do Órgão , Subpopulações de Linfócitos T/imunologia , Timo/imunologiaRESUMO
Little is known of the effects of nutritional deficiencies on lymphopoietic processes. Nevertheless, deficiencies in zinc adversely affect immune function causing thymic atrophy and lymphopenia in both humans and animals. Previous studies of the effects of zinc deficiency (ZD) on lymphopoiesis in adult mice indicated that a suboptimal intake of zinc caused a 50% or more depletion of the marrow of developing B cells. Thus, interference in the production of lymphocytes by ZD appeared to be a significant factor in the loss of host defence capacity. In the current study three-colour immunofluorescence phenotyping of early bone marrow B lymphocytes (B220+ immunoglobulin-) using flow cytometry demonstrated that a 27-day period of ZD caused a 50-70% decline in pre-B cells (B220+ CD43- immunoglobulin M (IgM)-) for moderate and severely zinc-deficient mice, respectively. Conversely, early pro-B cells (B220+ CD43+ 6C3-) and late pro-B cells (B220+ CD43+ 6C3+) exhibited little or no change in their distribution within the marrow. Indeed, the greater resistance of pro-B cells resulted in a 50% increase in the proportion of this subset within the B-cell compartment of the marrow as the deficiency in zinc advanced. Collectively, the data indicate that the B-cell compartment of the marrow is substantially altered by ZD and the stage specific sensitivity noted among early B cells may be related to chronically elevated levels of glucocorticoids present during ZD or other parameters that affect their survival and resistance to apoptosis.
Assuntos
Subpopulações de Linfócitos B/imunologia , Medula Óssea/imunologia , Zinco/deficiência , Animais , Peso Corporal , Diferenciação Celular/imunologia , Separação Celular , Feminino , Citometria de Fluxo , Imunofenotipagem , Camundongos , Camundongos Endogâmicos A , Tamanho do Órgão , Timo/patologia , Zinco/sangueRESUMO
Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0-6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosis in vivo. Mice were treated with a natural corticosterone (4-pregnene-11 beta,21-diol-3,20-dione) at levels detected during stress or malnutrition (93-180 micrograms serum cortisol/dl). At 4-12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.
Assuntos
Apoptose , Leucócitos , Sistema Linfático/imunologia , Fagocitose , Animais , Células da Medula Óssea/patologia , Corticosterona/farmacologia , Fragmentação do DNA , Humanos , Leucócitos/patologia , Sistema Linfático/citologia , Linfócitos/patologia , Camundongos , Tamanho do Órgão , Tonsila Palatina/patologia , Especificidade da Espécie , Baço/citologia , TimoRESUMO
Though lymphopenia is often noted in malnourished humans and rodents, little is known about the effects of suboptimal nutriture on lymphopoietic processes. Focusing primarily on cells of the B lineage in the marrow of young adult mice, a moderate degree of zinc deficiency (MZD) caused a 43% decline in the proportion of nucleated cells bearing B220 with a 91% decline noted among more severely zinc deficient mice (SZD). Early B cells (B220+Ig-) were highly sensitive to the deficiency, being barely detectable in SZD mice and reduced by almost 60% in MZD mice. Immature B cells (B220+IgM+IgD-) were similarly affected, declining 35% to 80% depending on the degree of the deficiency. In MZD mice, mature B cells (IgM+IgD+) exhibited moderate losses, being somewhat resistant. A more profound loss in this population was noted for SZD mice. Flow cytometric (FACS) scatter profiles indicated that zinc deficiency caused a sharp decline in the proportion of small nucleated cells which in the marrow are thought to contain a high proportion of developing lymphoid cells. There was a concomitant increase in large granular cells that paralleled a substantial increase in the proportion of nucleated cells bearing Mac-1 for both MZD and SZD mice. Given the dramatic depletion of cells of the B lineage in the marrow created by a deficiency in zinc, it is probable that disruptions in lymphopoietic processes in the marrow play a key role in the resulting lymphopenia observed in many types of malnutrition.
Assuntos
Linfócitos B/imunologia , Medula Óssea/imunologia , Linfopenia/imunologia , Zinco/deficiência , Animais , Subpopulações de Linfócitos B/imunologia , Peso Corporal , Feminino , Citometria de Fluxo , Luz , Linfopenia/etiologia , Camundongos , Camundongos Endogâmicos A , Tamanho do Órgão , Espalhamento de Radiação , Timo/patologiaRESUMO
Thymic atrophy and lymphopenia are immunological hallmarks of many forms of malnutrition including deficiencies in zinc. Extreme thymic atrophy (70-80%) along with a 50% loss of splenocytes in mice maintained on a zinc deficient diet (ZD) for 30 days suggested that the deficiency might be altering lymphopoiesis or the production of new lymphocytes by the bone marrow. As shown herein, mice who were marginally zinc deficient being 72-75% the body weight of adequately fed controls, exhibited a 50% decline in pre B-cells and a 25% decline in immature B-cells. The mature B-cells of the marrow appeared fairly resistant to effects of suboptimal zinc intake. Interesting, this pattern was similar to results obtained by treating bone marrow cells with levels of glucocorticoids analogous to those found in nutritionally deficient rodents. Furthermore, these same concentrations of steroids were shown to induce significant levels of apoptosis or cell death among pre and immature B-cells which accounted for their declining numbers subsequent to exposure to glucocorticoid. In order to better ascertain the potential role of glucocorticoids generated during zinc deficiency on lymphopoietic processes, adrenalectomies were performed in an attempt to remove glucocorticoids from the equation. Subsequently, adrenalectomized and sham operated mice were placed on a ZD or zinc adequate diet (ZA). Levels of steroids at the time of sacrifice were elevated six fold in non-adrenalectomized ZD mice compared to ZD adrenalectomized mice. Removal of the adrenal gland protected the thymus of ZD mice from atrophy and also provided substantial protection of lymphopoietic processes.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Apoptose/fisiologia , Glucocorticoides/fisiologia , Hematopoese/fisiologia , Linfócitos/fisiologia , Zinco/deficiência , Adrenalectomia , Animais , Células da Medula Óssea , HumanosRESUMO
A major role for polyomavirus middle T/small T antigens in viral DNA synthesis was uncovered by examining the replication of middle T/small T-deficient mutants (hr-t mutants). hr-t mutants in the A2 genetic background showed a 16- to 100-fold defect in genome accumulation relative to the wild type when infections were carried out in exponentially growing NIH 3T3 cells in medium supplemented with low levels of serum (< 2.0%). A proportional decrease in the level of viral early transcripts was also seen. The replication defect of the hr-t mutants was partially overcome in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. The defect was also alleviated by a duplication encompassing the alpha core enhancer domain that contains binding sites for the transcriptional activators PEA1/AP-1 and PEA3/c-ets. Such a duplication is present in all naturally occurring hr-t mutants and absent in the A2 strain. The effects of 12-O-tetradecanoylphorbol-13-acetate and alpha core duplication were additive but did not fully complement the absence of middle T/small T. In mixed infection competition experiments with two hr-t mutants, a genome that carried an alpha core duplication had a replication advantage (up to 17-fold) over a genome without duplication. This result demonstrates that one effect of the duplication is exerted directly at the level of DNA replication. The advantage of the duplication-bearing genome was established during the earliest stages of replication and was not further amplified in later rounds of replication. In the presence of middle T/small T, both genomes replicated to high levels and the advantage of the duplication-bearing genome was eliminated. On the basis of these results, we propose that factors that bind the alpha core domain (presumably PEA1 and PEA3) are present in limiting amounts in exponentially growing NIH 3T3 cells and play a crucial role in polyomavirus DNA replication. We further suggest that middle T and/or small T stimulates viral DNA replication by activating these factors. The fact that all middle T-/small T-defective hr-t mutants have evolved to contain enhancer duplications that encompass the PEA1 and PEA3 binding sites in the alpha core domain and partially restore their replication defect (A. Amalfitano, M. C. Chen, and M. Fluck, unpublished data) provides an adequate explanation for the fact that the importance of the role of the middle T and/or small T function in DNA replication has not been recognized previously. Much evidence is available in support of separate elements of this model.(ABSTRACT TRUNCATED AT 400 WORDS)