The aging of hematopoietic stem cells.

We have purified hematopoietic stem cells (HSCs) from the bone marrow of old mice and compared their properties to HSCs in young and middle-aged mice. Single, reconstituting HSCs (by limit dilution) from old and young mice exhibited indistinguishable progenitor activities in vivo. HSCs were five times as frequent in the bone marrow of old mice; however, HSCs from old mice were only one-quarter as efficient at homing to and engrafting the bone marrow of irradiated recipients. HSCs in young and middle-aged mice rarely were in the S/G2/M phases of the cell cycle, but HSCs in old mice were frequently in cycle. We speculate that the unexpected proliferation of HSCs in old mice might be related to the increased incidence of leukemia in old mice. HSCs change with age, but it is unknown whether these changes are determined intrinsically or caused by the aging of their environment.

In vitro studies on radiation lymphoid recovery of mouse spleen.

In vitro studies, utilizing an organ culture method were reported on mutual interactions between irradiated spleen, normal bone marrow, and thymus. It has been shown; (a) that singly isolated spleen explants were incapable of lymphoid regeneration, (b) thymus had no stimulatory effect on spleen regeneration, (c) bone marrow interacted synergistically with spleen leading to appearance of lymphoid cells which were not detected in singly isolated bone marrow or spleen, and (d) no stimulation of lymphopoiesis in bone marrow was conferred by thymus in the absence of spleen. The results are discussed in terms of possible mechanisms involved in lymphoid radiation recovery in vivo.

Reactivation in vitro of immunocompetence in irradiated mouse spleen.

The restoration of immunological competence to irradiated adult mouse spleen was studied using an in vitro assay system. Competence of the spleen to evoke a graft versus host reaction in vitro was lost upon sublethal irradiation (550 R). Regeneration of immune competence occurred when such spleens were grown for 2-3 days in the presence of thymus tissue, but not when grown in the presence of a variety of other tissues. This thymic activity was demonstrated to occur across an intervening Millipore filter barrier. When lethal doses of irradiation were used, reactivation of immune competence did not occur unless both thymus and bone marrow were present. In this experimental situation the bone marrow appeared to supply the immunocompetent cells.

Primary antibody response in organ cultures.

Specific antibody formation has been elicited in vitro following antigenic stimulation by either sheep (in a total of 472 of 875 cultures) or chick erythrocytes (in 65 of 135 cultures tested). The response was manifested by mouse spleen and lymph node explants whereas thymus cultures were inactive. The reaction has been characterized as a primary immune response in view of its kinetics as compared to defined primary and secondary responses, the effect of 2-mercaptoethanol on the antibodies formed, its subject to puromycin inhibition and its sensitivity to X-irradiation. Histological studies revealed preservation of the lymphoid cell populations throughout the entire experimental period.

Studies on characterization of the lymphoid target cell for activity of a thymus humoral factor.

The immune response to SRBC was measured in the spleens of adult thymectomized, total body irradiated mice injected with various combinations of thymus and bone marrow cells together with thymic humoral factor (THF). It was found that the number of plaque-forming cells was significantly increased when THF was given in vivo immediately after thymus cell administration or when thymus cells were incubated in THF before injection. On the other hand, bone marrow cells equally treated did not manifest any T cell activity, since THF-treated bone marrow cells were not able to substitute thymus cells in the system used. The results accumulated in the present experiments indicate, therefore, that the target cells for THF activity are thymus cells which acquire a higher T helper cell capacity after THF treatment.

Rejection of tumor allografts by mouse spleen cells sensitized in vitro.

This paper reports a model system of cellular immunity in which allosensitization of mouse spleen cells is induced in vitro. Allosensitization was achieved by culturing spleen cells upon monolayers of allogeneic fibroblasts. The ability of the spleen cells to inhibit the growth of tumor allografts in vivo served as a functional assay of sensitization. We found that unsensitized spleen cells or spleen cells sensitized against unrelated fibroblast antigens had no inhibitory effect on the growth of allogeneic fibrosarcoma cells when they were injected together into irradiated recipients. In contrast, spleen cells which were specifically allosensitized in vitro were found to be highly effective in inhibiting the growth of an equal number of allogeneic tumor cells. Several times more spleen cells from mice sensitized in vivo were required to produce a similar immune effect. This confirms the findings of previous studies which indicate that sensitization in cell culture can promote the selection of specifically sensitized lymphocytes. Preincubating sensitizing fibroblasts with allo-antisera blocked the allosensitization of spleen cells. This suggests that antibodies binding to fibroblasts may inhibit the induction of sensitization by competing with lymphocytes for antigenic sites. Mouse spleen cells which were able to recognize and reject tumor allografts in vivo were unable to cause lysis of target fibroblasts in vitro. Such fibroblasts, however, were susceptible to lysis by rat lymphoid cells sensitized by a similar in vitro method. These findings indicate that the conditions required for lymphocyte-mediated lysis of target cells may not be directly related to the processes of antigen recognition and allograft rejection in vivo.

Immunocompetence of spleen cells from neonatally thymectomized mice conferred in vitro by a syngeneic thymus extract.

Impaired immunological competence of spleen cells from neonatally thymectomized C57B1/6 young adult mice was apparent when these cells were tested in an in vitro graft-versus-host assay. Spleen cell inocula prepared from thymectomized mice did not induce enlargement of (C3H/eb x C57BI/6)F(1) newborn spleen explants, whereas the same number of cells from intact donors consistently initiated splenomegaly. Spleen enlargement was observed, however, when the explants were challenged by cells from thymectomized donors in the presence of syngeneic thymus extract, indicating that the spleen cells in suspension attained immunological competence under the influence of a non-cellular component of the thymus. Immunocompetence was also evident when the cells from thymectomized donors were first incubated with thymus extract for 1 hr and subsequently tested for reactivity. Cells from the same thymectomized donor mice exposed in parallel to extracts from syngeneic spleen or mesenteric lymph node at an equivalent protein concentration did not initiate a graft-versus-host response. These experiments demonstrate that immune reactivity in the graft-versus-host response involves activation of lymphoid cells by a humoral factor of the thymus acting directly upon these cells.

Autosensitization in vitro.

Autosensitization of rat or mouse lymphoid cells against syngeneic fibroblast antigens was induced in cell culture. Rat lymphoid cells autosensitized by this method were able to produce immunospecific lysis of syngeneic target fibroblasts in vitro or GvH reactions in newborn rats. Autosensitized mouse spleen cells mediated similar GvH reactions when injected into newborn mice. The nature of the system used to induce immunity in vitro appears to argue against the possibility that lymphocytes capable of reacting against self-antigens could arise by mutation in cell culture. Hence, it is likely that cells potentially reactive against self-antigens preexisted in the lymphoid cell donors. The ability of autosensitized cells to mediate immune reactions in vivo suggests that the immunogenic self-antigens present on sensitizing fibroblasts also were accessible in the intact animals. Loss of natural self-tolerance in vitro, therefore, can be explained most simply by the existence of lymphocytes which are reversibly tolerant to self. Hence, ontogenic elimination of potentially self-reactive cells may not be the only basis for natural tolerance. Regulatory mechanisms, such as antigen excess, may have to function in vivo to prevent differentiation of self-tolerant lymphocytes. These regulatory mechanisms appear to be annulled in the cell-culture system. The present system thus may offer a new approach to studies of tolerance and regulation of cellular immunity.

In vitro induction of a primary response to the dinitrophenyl determinant.

Primary antibody response against the dinitrophenyl group has been elicited in vitro after the stimulation of normal mouse spleen explants with 2,4-dinitrophenyl (DNP)-hemocyanin or alpha-DNP-poly-L-lysine (PLL). Antibodies were detected in the culture medium by the inactivation of DNP-T4 phage. The specificity of the reaction was manifested by the lack of the capacity of the medium to inactivate the unmodified bacteriophage and by the inhibition of the inactivation of DNP-T4 with DNP-lysine.

Evidence-based roads to the promotion of health in old age.

The increase in life expectancy, along with the accompanying ongoing increase in the proportion and absolute numbers of nonagenarians and centenarians have set forth the curiosity regarding the question of the quality of health in very old age. Studies on that issue have pointed to the fact that the very old people are actually healthier than originally predicted on the basis of the earlier studies on aging. Current efforts are thus invested in elucidating the possible basis of health in the very old people, as well as better understanding of potential causes of frailty and common diseases in old age. This review recounts on the various aspects underlying evidence-based recommendations for healthy life in old age. We focus on the genetic and non-genetic bases of aging and longevity, and the various directions towards the promotion of health, both via avoiding, or eliminating risk factors and deleterious effects, as well as conducting healthy lifestyle - in terms of proper nutrition and physical exercise. Next, we touch upon preventive medicine, particularly as related to vaccination, with a note also on the need for a reasonable use of medications. In addition, we report about the developing area of regenerative medicine and its potential in relation to the prevention of damage and possible strategies towards tissue repair in cases of age-related degenerative processes.

Selective accumulation of lymphocyte precursor cells mediated by stromal cells of hemopoietic origin.

Thymocytes were propagated in long-term cultures supported by stromal cells of both bone marrow and thymus origin. Interleukin 2 (IL-2) supplementation augmented the cell yield and allowed detailed phenotype analysis. Within 2-3 months of culture a cell population was selected in which the expression of Thy-1 antigen persisted, CD4 and CD8 antigens gradually declined, and Pgp-1 antigen, found on less than 5% of fresh thymocytes, was strongly increased. This cultured cell population (Thy-1.2 origin) contained no detectable spleen colony-forming units (CFU-S) but efficiently repopulated the thymus of Thy-1.1-irradiated congenic mice, indicating the precursor T-cell nature of the population. Upon removal from the stroma, the T cells exhibited poor cytotoxicity towards syngeneic tumor cells. Further propagation with IL-2 in the absence of stroma resulted in the acquisition of cytotoxic ability. Replacement of the horse serum used in the above experiments with fetal calf serum resulted in accumulation of cells expressing B220 antigen. This experimental model provides the means to maintain lymphocyte precursor cells in long-term culture and to further study their differentiation in the absence of stroma, both in vitro and in vivo.

Selective adhesion of immature thymocytes to bone marrow stromal cells: relevance to T cell lymphopoiesis.

We investigated the interactions between the bone marrow microenvironment and T cell populations at different stages of maturation. Thymocytes were seeded onto confluent layers of bone marrow stromal cell lines (MBA-13 or 14F1.1). Within a few hours two main thymocyte populations were observed; one remained in the liquid phase and the other adhered to the stromal cells. After 24 hours of culture, most of the adhering cells expressed the phenotype of the precursors, double negative (DN) CD4-CD8-, or of immature thymocytes, double positive (DP) CD4+CD8+. The number of adhering DN cells did not change during the time of the culture, whereas that of the DP declined. The CD4+CD8- or CD4-CD8+ cells did not adhere to any significant extent. The expression of CD3 antigen on adherent thymocytes was lower than that on nonadherent ones. Sorted thymocytes at a high level of purification (>96%) were cultured over stromal layer and, after 24 hours, 60% of the DN or 22% of the DP cells were found to adhere to the stroma. The culture medium was replaced every 24 hours or after 48 hours; no significant change was noted in the number of adhering DN and DP cells. The reappearance of immature T cells in the liquid phase suggested proliferation of this cell type. Thus, early thymocytes, phenotypically characterized as DN and DP, preferentially adhere to bone marrow stromal cells. This in vitro phenomenon may represent the function of the BM stroma as an extrathymic site of T cell lymphopoiesis.

Differential effects of CD4+ and CD8+ cells on lymphocyte development from human cord blood cells in murine fetal thymus explants.

The possibility that mature lymphocytes play a role in the regulation of human T cell development was studied in the experimental model of fetal thymus organ cultures (FTOC), by reconstituting lymphocyte-depleted murine fetal thymus (FT) lobe with cells isolated from human umbilical cord blood (CB). Cultures were incubated with human cytokines (IL-7, FLT-3 ligand and Steel Factor), or remained untreated. When CD4+, or CD8+ CB cells, were co-cultured with FT explants, they expanded and maintained their original phenotypic markers, with no significant effect of the cytokines. Cultures of human hematopoietic stem cells (CD34+) gave rise to CD4+CD8- cells, which were mainly CD3-, with no indication of further intermediate developmental stages. However, a limited number of CD4+CD8+ (double positive [DP]) cells were detected when the CD34- cells were co-cultured with CD4+ cells from the same CB samples. In contrast, FT with unseparated CB cells resulted in the different CD4/CD8 subsets, and their numbers increased in the presence of cytokines. The appearance of DP cells depended on the presence of either CD4+ or CD8+ cells in the cultured CB samples. Hence, DP cells were not detected when the CB was depleted of CD4+ and CD8- cells ("depCB") before culture, and they appeared when depCB were co-cultured with either CD4+ or CD8+ cells. In contrast, CD4+ cells inhibited the development of CD8+CD3+ cells, and this was most pronounced in the absence of the cytokines. There was no symmetrical down-regulatory effect of CD8+ cells on the development of CD4+CD3+ cells. Addition of IL-15 to the cytokine mixture led to an increased proportion of CD56+ cells in cultures of CD34+ cells. The presence of CD4+, and not CD8+ cells, interfered with this process. Our results thus imply differential effects of CD4+ and CD8+ cells on thymocytopoiesis.

AlphaMUPA mice: a transgenic model for increased life span.

AlphaMUPA is a line of transgenic mice that, compared with their wild type (WT) counterparts, spontaneously eat less (approximately 20%) and live longer (average approximately 20%), thus resembling dietary-restricted (DR) mice. Here, we show that body temperature was significantly reduced in alphaMUPA compared with WT throughout a wide range of ages. Plasma corticosterone was significantly higher in young alphaMUPA compared to young WT; however, it significantly declined in aged alphaMUPA, but not in aged WT. In addition, age-associated thymus involution occurred in alphaMUPA as it did in WT. Thus alphaMUPA mice appear to largely resemble, but also to somewhat differ from diet-restricted animals. We also report on four new transgenic lines that, like alphaMUPA, produced in the brain the mRNA that encodes the extracellular protease urokinase (uPA); however, transgenic uPA expression was most extensive and widespread in the alphaMUPA brain, where it also occurred in the hypothalamus. AlphaMUPA was also the only line that ate less, but also showed another characteristic, high frequency leg muscle tremor seen only at unstable body states. We hypothesize that transgenic uPA in the brain could have caused the alphaMUPA phenotypic alterations. Thus alphaMUPA offers a unique transgenic model of inherently reduced eating to investigate the homeostatic state of delayed aging at the systemic and single-cell levels.

Immune reactivity during aging. I. T-helper dependent and independent antibody responses to different antigens, in vivo and in vitro.

The immunological status during aging was assessed by measuring the antibody response of the long-lived (C3H/eb X C57Bl/6J)F1 mice to various antigens in vivo and in vitro. In vivo, a decrease in antibody production to DNP and NIP haptenic determinants coupled on to BGG, as well as the response to SRBC, was observed. The decline was more pronounced in the IgG as compared to IgM antibodies. The results were recorded when various parameters such as antigen dose and kinetics of the response, were considered. Reduction of the antibody response was also noted when PVP was employed as immunogen. Similar results were noted when the responses to SRBC and DNP--polylysine were induced and followed in spleen organ cultures. In all of these experimental systems, the peak response was observed in mice 6--12 months old. From then on a gradual decrease was manifested, mice 30--36 months old producing significantly low responses. The results demonstrate that decrease in antibody production is expressed in the isolated spleen tissue in the same manner as in the intact animal. Furthermore, they were interpreted as indicating that the lesion may be at the T helper and the B cell compartments.

Immune reactivity during aging. II. Analysis of the cellular mechanisms involved in the deficient antibody response in old mice.

Analysis was made of the cellular basis of decline in the antibody immune response with advancing age. Employing the cell transfer system, it was found that spleen cells of aged mice have a limited capacity to react to SRBC when transferred into young irradiated recipients. Partial reconstitution was achieved when thymus cells from young untreated donors were applied together with the spleen cells, suggesting that deficiency in T helper cell function is responsible in part for the limited response. Similar studies using the T helper-independent antigen PVP also resulted in low levels of antibodies in the irradiated recipients. Treatment of the spleen cells with anti-theta serum before transfer did not lead to an increased response. Hence, it is suggested that the reduced response is related to limited function in the B cell compartment, and not to function of T suppressor cells. Transfer of bone marrow cells from aged into young irradiated recipients indicated that cells capable of differentiation into antibody-producing cells are available during aging. It is thus maintained that the defect at the B cell level is of a developmental nature.

Viscosity of lymphocyte plasma membrane in aging mice and its possible relation to serum cholesterol.

Lymphocyte membrane fluidity was examined in aged mice and characterized as a qualitative and effective change which takes place in the aging process. Fluorescence polarization of diphenylhexatriene-labelled mouse spleen cells is substantially higher in cells from old mice (20--36 months) than young mice (2--7 months). A similar difference was also observed with isolated plasma membranes from spleen cells of old and young mice. The overall estimate is that the lipid microviscosity in the lymphocyte plasma membrane from old mice is about 20% higher than that of young mice. The cholesterol/phospholipid ratio determined for the isolated plasma membrane preparations was 0.68 and 0.9, respectively, which is probably the main cause for the difference in membrane viscosity. An elevated cholesterol/phospholipid ratio was also observed in the blood serum of old mice. It is plausible that the source of excess membrane cholesterol in the old mouse lymphocytes originates in the high serum cholesterol.

Thymocyte progenitors and T cell development in aging.

Dysfunction of T lymphocytes in aging has been causally related to a gradual loss of the thymic microenvironmental function. However, in view of the fact that T cells are generated from bone marrow-derived stem cells that settle in the thymus, we have investigated the possibility that aging effects on the bone marrow have an impact on T cell development. Our approach was based on seeding of bone marrow cells, from young and old mice, onto lymphoid-depleted fetal thymus explants, and examining the patterns of T lymphocyte development under organ culture conditions. The results indicate multifactorial effects of aging, on pre-thymic and intra-thymic development processes, as well as on feedback regulation by mature T cells.

Immune reactivity during ageing. III. Removal of peanut-agglutinin binding cells from ageing mouse spleen cells leads to increased reactivity to mitogens.

Spleen cells from ageing (24--30-month-old) mice, manifesting low response to concanavalin A (Con A) and phytohemagglutinin (PHA), were separated by peanut agglutinin (PNA) into agglutinable (PNA+) and unagglutinable (PNA-) fractions. The PNA+ cells were found to suppress the response of young (2--3-month-old) mouse spleen cells to the mitogens Con A and PHA. On the other hand, PNA- cells of the ageing mice expressed a response to these mitogens, at levels higher than those of the unseparated cells. Re-mixing of the PNA- and PNA+ cells of the ageing mouse spleens at various proportions indicated that the PNA- cells are indeed suppressible by the PNA+ cells. Anti Thy-1.2 treatment to the PNA+ fraction suggested that this suppression was not exerted by T cells. It thus appears that at least part of the reduced lymphoid cell function in ageing is related to an effect of suppressor cells, interfering with the expression of available reactive cells.

The bone marrow as an effector T cell organ in aging.

The idea that the bone marrow (BM) might function as a T cell effector organ and might constitute a compensating system to the decreased T cell compartment in aging was examined by carrying out an analysis of T cell functions in this tissue. The Thy1+ cells, which were found to increase in their proportions with age in the BM, were sorted out from the BM of young and old mice by flow cytometry and their proliferative response to Concanavalin A (conA) stimulation was measured. The sorted Thy1+ cells responded to conA at levels comparable to those of splenocytes, with the old BM showing a significantly lower response than the young. To determine whether cells of the intact BM behave in a similar pattern to the sorted cells, we measured the responses induced by conA in unseparated BM cells of both age groups. The results showed that the patterns of proliferative response in the intact BM cells were different than those observed in splenocytes and in the Thy1+ sorted cells. Hence, cells of the old BM manifested initially higher levels of proliferation preceding that of the young BM, yet the response was apparent for a shorter duration. When we measured the conA induced proliferation of the intact BM cells in the presence of colchicine, the number of BM cells entering the first mitotic cycle was higher in the old. Thus, it seems that there are more effector T cells in the old BM, yet their response to stimulation is of shorter duration. This conclusion was also supported by the assessment of cytotoxic T lymphocytes precursor (CTLp) frequency and of CTL function of the BM. CTLp was higher in the old BM following 3 days of incubation and lower following 7 and 9 days of incubation. The old BM cells showed a reduced CTL response at the proliferative phase when stimulated for 4-7 days, yet their effector cell reactivity was either equal or more efficient than the young. To determine whether some of the differences between the two age groups were due to regulatory effects of the BM microenvironment, BM cells from young and old donors were admixed with young mouse splenocytes and stimulated with conA. The conA induced response was enhanced up to tenfold under these conditions yet to the same extent in both age groups. Thus, it appears that the BM has the capacity to function as a T cell effector organ.(ABSTRACT TRUNCATED AT 400 WORDS)