Lymphocyte Proliferative Capacity Research Articles

The biology of Stat4 and Stat6.

IL-4 and IL-12 are cytokines that are important regulators of the proliferation, differentiation and functional capacity of lymphocytes. STATs (signal transducers and activators of transcription) are transcription factors that provide a direct link between the cytokine receptors and cytokine induced gene transcription. Stat6 and Stat4 are two STAT family members that specifically mediate signals that emanate from the IL-4 and IL-12 receptors, respectively. Recently a great deal of progress has been made in understanding the specific roles that Stat6 and Stat4 play in lymphocyte function through in vitro as well as in vivo studies using Stat6 and Stat4-deficient mice. This report will summarize and describe the recent advances made in understanding the activation and regulation of Stat6 and Stat4 as well as their roles in the development of an immune response. Oncogene (2000).

White cell apoptosis in packed red cells.

After the removal of the buffy coat, packed red cell (RBC) transfusion units still contain white cells that may undergo apoptosis as a result of storage conditions (1-6 degrees C). The aim of the present study was the evaluation of this phenomenon in view of the possible influence it may have on febrile nonhemolytic transfusion reactions. Three independent methods (microscopy, DNA electrophoresis, and cytometry) were used to evaluate apoptosis in white cells present in 13 RBC units. Of these units, 10 had been collected into CPD/saline-adenine-glucose-mannitol and 3 into CPDA-1; each bag was split in two parts, one of which was irradiated. RBCs were stored at 1 to 6 degrees C, and samples were periodically withdrawn for study. The proliferative capacity of stored lymphocytes was evaluated after phytohemagglutinin stimulation and tritiated thymidine incorporation. Apoptosis was found to occur in both granulocytes and lymphocytes, starting from the first 48 to 72 hours of storage. The choice of the anticoagulant-preservative solution and the effect of irradiation did not influence the amount and the timing of the apoptotic phenomenon. Lymphocyte proliferative capacity was found to decrease sharply with storage time. Conditions of storage in RBCs induce consistent apoptosis in residual white cells. The possible clinical implications of the relationships between apoptosis and the induction of biologic response modifiers (that may cause interleukin-mediated febrile non-hemolytic transfusion reactions) and between apoptosis and immune reactions remain to be elucidated.

Long-term melatonin supplementation does not recover the impairment of natural killer cell activity and lymphocyte proliferation in aging mice

In this study we evaluated the effect of long-term melatonin (MEL) treatment on the cytotoxic activity and number of natural killer (NK) cells and the proliferative response of spleen lymphocytes to phytohemagglutinin (PHA) or interleukin-2 (IL-2) in old mice. Seventeen-eighteen month-old Balb/c mice were supplemented with MEL (40–50 μg/day/mouse) and sacrificed after eight months. The MEL supplementation was unable to recover the low levels of both endogenous and IL-2-induced NK cell activity found in old untreated mice Also the NK cell number was unaffected by MEL treatment. The spleen lymphocyte proliferative response to both PHA and IL-2 was not different in old MEL-treated compared to old untreated mice. These results indicate that long-term MEL supplementation does not recover the age-related deterioration of NK cell activity and lymphocyte proliferative capacity.

A rapid qualitative method to assess in vitro immunobarrier competence of pancreatic islets containing alginate/polyaminoacidic microcapsules.

A quick method for the qualitative evaluation of immunoisolatory properties associated with islet-containing alginate/poly-L-ornithine (AG/PLO) microcapsules is described. In particular, we examined a new AG/PLO coherent microcapsule (CM) prototype that was recently developed in our laboratory, although the procedure could be applicable to other capsule types as well. We observed no binding of immunoglobulins (Ig) contained in islet cell antibody (ICA)-positive human sera (> 60 Juvenile Diabetes Foundation units, JDT U) to pig islets, enveloped within AG/PLO CM, under indirect immunofluorescence examination. Also, CM were shown to inhibit human lymphocyte proliferative capacity fully, as assessed by the 3H-thymidine incorporation rate, in in vitro mixed xenogeneic pig islet/human lymphocyte co-cultures. These results provided us with a preliminary method to attempt standardization of basic physical/chemical properties which should be associated with an immunoisolatory membrane for islet allo/xenograft immunoprotection.

EFFECT OF CARNITINE DEPRIVATION ON LYMPHOCYTE PROLIFERATION IN THE NEONATAL PIG. 1882

Carnitine has been reported to have immunomodulatory effects (De Simone, 1994 AIDS 8:655-660). Neonates are at increased risk for carnitine deficiency if intake is inadequate. To investigate possible effects of carnitine deprivation on lymphocyte function, blood samples were obtained from 2 to 3 week-old piglets that received parenteral nutrition with (CARN+, n=17) or without (CARN-, n=16) carnitine, or were sow-fed (SF,n=13). Triplicate cultures containing 200,000 lymphocytes isolated by ficoll hypaque centrifugation were incubated with phytohemagglutinin (PHA: 0, 50, 100, 200μl/ml) for 48 hours before being pulsed with 3H-thymidine for 20 hours. Cells were harvested on filter strips for determination of radioactivity incorporated into DNA. In a second series, lymphocytes were similarily harvested and incubated with PHA and L-carnitine (0,5,50,500μM).Results: CARN - animals exhibited decreased mean carnitine plasma levels compared with CARN + or SF animals. Large interindividual variations were seen, but no differences in mean basal 3H-thymidine uptake were found. PHA stimulated DNA synthesis in all groups. The stimulation index (SI:[CPM + PHA]/ basal CPM) was inversely correlated with basal CPM. However, no differences were found among the 3 groups. L-carnitine in vitro had a variable effect on non-stimulated 3H-thymidine uptake, but enhanced PHA-stimulated increases of SI in CARN+ animals. The findings suggest that nutritional carnitine deprivaton does not significantly affect lymphocyte proliferative capacity. (Funded by USPHS HD29273 and USPHS K14 HL03144)

Effects of interferon-alpha treatment on neutrophil oxidative metabolism, lymphocyte proliferation and monocyte HLA class I antigen expression in patients with chronic hepatitis C.

Polymorphonuclear cell (PMN) oxidative metabolism, lymphocyte polyclonal proliferation and monocyte HLA class I antigen expression were evaluated at different intervals of time in patients with chronic hepatitis C (CH-C) subjected to a 6 month interferon alpha (IFN-alpha) treatment and divided into Responder ('R') and Nonresponder ('NR') subsets according to clinical outcome. Before therapy, all subjects exhibited multiple immune alterations even if to a different extent between 'R' and 'NR' subsets: an elevated superoxide anion (O2-) generation by suspended PMN, a failure to further increase neutrophil oxidative responsiveness under adherence conditions, an augmented phytohaemagglutin-induced lymphocyte proliferative capacity and an enhanced HLA class I antigen expression on CD14+ cells. IFN-alpha administration gave rise to a modulation of oxidative response in 'R' group only, since these individuals displayed an O2- release by suspended and adherent PMN which fell within normal values. At the same time, a decrease of lymphocyte proliferation occurred in both groups of patients during IFN-alpha therapy, even if it reached statistical significance in 'R' group only. Finally, a more marked difference between 'R' and 'NR' individuals was noted in terms of HLA class I antigen induction on CD14+ cells at the end of therapy, as a consequence of a reduced expression of these structures in 'NR' subjects. Altogether, these findings suggest the occurrence of a strict relationship between immunoresponsiveness and IFN-alpha induced therapeutical effects in CH-C patients.

Enhancement of in vitro nonspecific immune functions by African plant extracts

AbstractThe in vitro immunomodulatory activities of extracts from African medicinal plants have been studied. Certain extracts exerted enhancing effects in a concentration dependent manner on immune cells from three different strains of mice. Among these were an enhancement of phagocytic activity of peritoneal macrophages and tumoricidal potential of macrophages, and stimulation of the oxidative burst of macrophages and granulocytes. In contrast, the proliferative capacity of lymphocytes was only marginally affected. Enhanced macrophage and granulocyte activation was also detected using leucocytes of the LPS nonresponder strain C3H/HeJ. From the latter it can be concluded that the observed stimulatory effects of the plant extracts in vitro were not due to contamination with lipopolysaccharides.

Energetics of Lymphocyte "Burnout" in Late Sepsis: Adjuvant Treatment with ATP-MgCl2 Improves Energetics and Decreases Lethality

Although it is known that decreased high-energy phosphates contribute to organ dysfunction following shock, it remains unknown whether changes in lymphocyte energetics contribute to the profound immune dysfunction that occurs in late septic shock. Moreover, while studies have shown that ATP-MgCl2 treatment after hemorrhagic shock improves tissue ATP levels and organ function, it remains unknown whether lymphocyte high-energy phosphates and immune functions are similarly affected by this agent after sepsis. To study this, sepsis was induced in C3H/HeN (endotoxin sensitive) mice by cecal ligation and puncture (CLP) and they were then treated intraperitoneally with ATP-MgCl2 or saline vehicle. Sham animals received laparotomy, but not CLP. Splenic lymphocytes were harvested 24 hr after treatment and ATP levels were determined by ultraresolution 31 P NMR. Lymphocyte proliferative capacity was determined by [3H]-thymidine incorporation following mitogenic stimulation. Host survival was assessed following CLP with and without ATP-MgCl2 treatment. Prolonged sepsis caused a significant decrease (↓ 67 ± 12% vs Sham) in lymphocyte ATP levels which were correlated with decreased lymphocyte proliferative capacity in response to mitogenic stimulation (64 ± 17 × 103 vs. 232 ± 43 × 103 counts per minute (cpm) in Sham; P < 0.05). Treatment with ATP-MgCl2 at the onset of sepsis significantly increased lymphocyte ATP levels (↑ 32 ± 15% vs CLP) and proliferative response to mitogenic stimuli (218 ± 37 × 103 cpm, CLP/ATP-MgCl2; P < 0.05). Improved lymphocyte function in this group correlated with a significant increase in overall survival (20% CLP vs 70% CLP/ATP-MgCl2; P < 0.05). We conclude that: (1) decreased lymphocyte ATP levels may be the cause of defective lymphocyte proliferative capacity in late sepsis, (2) adjuvant treatment with ATP-MgCl2 improves both lymphocyte ATP levels and lymphocyte proliferative capacity, and (3) intraperitoneal ATP-MgCl2 decreases lethality from sepsis.

Phenomena of Spontaneous Proliferation, Differentiation, and Apoptosis in Primary Culture of Leukemic Lymphocytes

The proliferative capacity of lymphocytes from peripheral blood of bovine with chronic lymphocytic leukemia (CLL) in vitro was investigated. We have shown earlier that CLL cells spontaneously proliferate in serum-free medium in the absence of added growth factors and mitogenic stimulation; autocrine growth factors provide the growth-initiating signal for CLL cells. The results of the present study showed that bovine serum albumin or fetal calf serum greatly enhanced the number of CLL cells incorporating [3H]thymidine. Although some CLL cells proceeded through more than one cell cycle, proliferation of CLL cells in culture was temporary. On the other hand, it was shown that CLL cells differentiated spontaneously in culture. This differentiation was characterized by the appearance of plasmacytoid cells possessing cytoplasmic immunoglobulins that coincided with the cessation of cell proliferation. Moreover, together with spontaneous proliferation and differentiation, the phenomenon of programmed cell death (apoptosis) was found, as was evidenced by the appearance of apoptotic bodies as well as DNA fragmentation. The findings indicate that the loss of proliferative potential of CLL cells in culture may be a consequence of their differentiation and/or apoptosis in vitro. CLL cells, with an autotrine growth mechanism, spontaneous differentiation, and apoptosis in vitro, provide a new model system for studies of the relationship between cellular proto-oncogene expression and inhibition of growth and/or induction of differentiation.

The effect of aging on laboratory values in dogs.

The main objective of this study was to examine age-associated changes in laboratory values in dogs in order to establish standard values for dogs of a certain age. These standard values could serve to judge the health status of individual dogs and possibly estimate their remaining life expectancy. Thus, haematological and immunological parameters, plasma constituents, kidney characteristics and the capacity of homeostasis were determined in German shephard dogs (0.5-13.5 years) and beagles (4-9 years). Significant changes were noted. We detected that the number of white blood cells declines with age. The change in a number of organ specific enzymes indicates organ involution and increased cell death. Increased plasma glucose levels and a decreased glucose tolerance point to a diminished sensitivity of tissue to concentrations change due to a decrease in organ production and changes in the immune system, which are also reflected in an impaired lymphocyte proliferation capacity. These factors lead to an increased blood sedimentation rate. Cortisol increases might reflect a reduced stress resistance. Age-related decreases in aldosterone concentration and PAH-clearance were also found.

Effect of cyclosporin A and zidovudine on immune abnormalities observed in the murine acquired immunodeficiency syndrome.

Two therapeutic modalities, zidovudine (targeting retroviral replication) and cyclosporin A (targeting immunopathologic consequences of retroviral expression) were evaluated in a murine model of AIDS. In previous studies, cyclosporin A treatment (40 or 60 mg/kg/day) before and after infection with LP-BM5 murine leukemia viruses protected against the development of immunodeficiency disease. The present study extends these findings. First, a low dose of cyclosporin A (20 mg/kg/day) was ineffective, and treatment initiated 5 days after infection did not protect against virus-induced lymphoproliferation and hypergammaglobulinemia. Second, zidovudine added to drinking water (0.1 mg initiated 5 days after infection and continued for 8 weeks) was more effective than 0.2 mg/mL given day 5-12 after infection. This treatment reduced lymph node size, disease severity as determined histologically, retrovirus-induced gp70 expression, and IgE (but not IgM and IgG) levels. Third, combined treatment had an additive, protective effect on lymphocyte proliferative capacity. This successful dual therapeutic strategy in a mouse model has potential applicability for similar approaches in treating human immunodeficiency virus infection.

Improvement in the proliferative capacity and natural killer cell activity of murine spleen lymphocytes by thyrotropin.

In the present study we investigated the effect of thyrotropin (TSH) on both the proliferative capacity and the natural killer (NK) cell activity of murine spleen lymphocytes. It was found that TSH at various concentrations significantly increased the proliferative response of mouse lymphocytes to both concanavalin A (Con A) and phytohemagglutinin (PHA). This increase was particularly evident when suboptimal concentrations of mitogens were used (40-50% increase). The administration to cell cultures of TSH alone could not induce a significant stimulation of proliferative capacity. In order to provide a better knowledge about the mechanism by which TSH improved the mitogen-induced lymphocyte proliferation, the effect of the pituitary hormone on lymphocytes directly stimulated with recombinant interleukin-2 (RIL-2), was examined. It was observed that there was a great increase in IL-2-induced lymphocyte proliferation by TSH. The improvement in proliferative capacity of lymphocytes was particularly evident by using suboptimal rIL-2 concentrations (25-30% increase). The studies carried out on the cytotoxic activity of NK cells showed that TSH was able to significantly increase the IL-2-induced NK cell activity without modifying the basal levels of cytotoxicity. The results support the immunoregulatory role of TSH and contribute towards understanding the mechanisms of interaction between neuroendocrine and immune systems.

Soluble IL-2 receptor as an agent of serum-mediated suppression in human visceral leishmaniasis.

In visceral leishmaniasis (VL), patient's lymphocytes are not activated by leishmania Ag stimulation, and their sera exhibit a potent nonspecific suppressive effect on the responses of normal lymphocytes. Sera were obtained from 33 VL patients, eight patients with subclinical VL, and from 27 normal volunteers. Only sera from VL patients markedly reduced Con A-induced lymphocyte proliferative responses, as well as IL-2 or IFN-gamma production by normal lymphocytes. Addition of exogenous human rIL-2 to cultures containing VL patient sera partially reversed the normal lymphocyte proliferative capacity and restored IFN-gamma production. This phenomenon was consistent with the presence of greatly elevated levels of soluble IL-2R (sIL-2R) in VL patients' sera (4299 +/- 2351 U/ml), well above those of normal sera (180 +/- 94 U/ml), or of sera from patients with subclinical leishmania infection without immunosuppression (1002 +/- 281 U/ml). Furthermore, the removal of sIL-2R reduced VL serum suppressive activity as evaluated by effects on IL-2 and on IFN-gamma production. These data suggest the participation of high levels of sIL-2R in the serum-mediated suppression in VL.

A further exploration of the relationships between immune parameters and the HPA-axis activity in depressed patients

In order to investigate the relationship between the immune apparatus and the hypothalamic-pituitary-adrenal (HPA)-axis activity in depressed patients, we measured in vitro lymphocyte responses to the mitogens Phytohaemagglutinin (PHA), Pokeweed (PWM) and Concanavalin A (Con A) and 8 a.m. baseline cortisol values in plasma, free cortisol excretion in 24 h urine (UFC), basal and post-dexamethasone beta-endorphin values. Major depressed patients with melancholia/psychotic features exhibited a significantly lower mitogen-induced blast transformation as compared to minor and simple major depressed patients. The lymphocyte responses to the three mitogens were significantly inversely related to baseline cortisol values and postdexamethasone beta-endorphin values. The proliferative capacity of lymphocytes to stimulation with PHA and PWM was significantly and positively related to UFC excretion. Up to 45% of the variance in the immune-responses to the mitogens was explained by the baseline cortisol, post-dexamethasone beta-endorphin and UFC values.

Modulating Effects on CD25 and CD71 Antigen Expression by Lectin-Stimulated T Lymphocytes in the Elderly

During the last few years, several observations outline that the impaired T lymphocyte proliferative capacity in the elderly is due to a reduced interleukin 2 (IL-2) release. To further investigate the activation process during lectin stimulation, aged peripheral blood mononuclear cells (PBMC) were stimulated with phytohemagglutinin (PHA) and assessed for CD25 (IL-2 receptor) and CD71 (transferrin receptor) expression at different intervals of time. Our results provided evidence for a significant decline of both structure induction, above all in the later phase of culture. Indomethacin (INDO) treatment gave rise to an enhancement of CD71 antigen expression only, while prostaglandin E2 (PGE2) supplementation to culture media further decreased either CD25 or CD71 receptor induction. Interferon (IFN)-alpha and IFN-gamma treatment failed to modulate the frequency of CD25+ and/or CD71+ cells. Finally, the expression of CD71 receptor was increased by deferoxamine supplementation, this suggesting a partial involvement of iron overload in the depressed function. Although further studies are required to evaluate at a molecular level the decreased antigen expression, these findings indicate that several mechanism are involved in the elderly-related decline of T lymphocyte activation structures during lectin stimulation.

Effect of age on lymphocyte proliferation

The relationship of lymphocyte proliferative capacity to age was studied using lymphocytes from neonates, from young adults aged 20–30 and from healthy individuals aged 70–90. Mass cultures expanded exponentially and eventually died after a final expansion of 10 19–10 52. They therefore showed a Hayflick effect, but in contrast to reported findings for other cell types there was no relationship between age and the magnitude of the final expansion. Cytogenetic and molecular studies showed that monoclonality developed in all mass cultures. Study of individual clones also showed exponential growth followed by cessation. The magnitude of the expansion, 10 4–10 35, was substantially less than that observed for mass cultures, but was related to age. We conclude that lymphocytes have a heterogeneous proliferative potential, that the overall proliferative potential declines with age but that rare cells having extended proliferative potential continue to be present into old age. The development of monoclonality during the course of mass cultures has implications for the interpretation of findings from such cultures since observations drawn from the later stages of culture reflect the properties of rare cells having high proliferative potential and do not necessarily reflect the properties of the overall population.

Expression of asialo GM1 on a subset of adult murine thymocytes: histological localization and demonstration that the asialo GM1-positive subset contains both the functionally mature and the proliferating thymocyte subpopulations.

A small population (10 to 14%) of adult murine thymocytes expresses the glycolipid asialo GM1 (aGM1). Flow cytometric analysis of the aGM1+ cells present in thymus demonstrates the expression of a mature or medullary phenotype by 50% of the aGM1+ cells. Analysis of cytotoxic T lymphocyte precursor activity, proliferative capacity, and IL 2 production displayed by aGM1+ and aGM1- thymocyte fractions isolated by cell sorting indicates that these functional compartments of the thymus are contained within the aGM1+ subset. The aGM1+ population also contains virtually all mitotically active thymocytes, as measured by incorporation of bromodeoxyuridine. The immature IL 2 receptor-bearing thymoblasts are also included in the aGM1+ population. Immunohistochemical labeling of thymic tissue sections reveals that the majority of aGM1+ cells are located in the medulla. Clusters of aGM1+ cells are found scattered throughout the cortical and subcapsular areas. The aGM1+ population therefore contains the functionally mature thymocytes as well as some immature thymocytes, particularly those that are mitotically active. It is suggested that the aGM1+ subset of thymocytes represents those cells that are mature or actively maturing. This hypothesis is discussed in the context of current concepts of intrathymic T cell differentiation pathways.

Suppression of lymphocyte proliferative responses by sera from patients with American visceral leishmaniasis.

We examined the effect of sera from 11 patients with American visceral leishmaniasis on mitogen-driven lymphocyte proliferative capacity. All sera inhibited lymphocyte proliferation of patients' peripheral blood mononuclear cells (PBMC) when stimulated by either phytohemagglutinin, Concanavalin A or pokeweed mitogen. Serum was also strongly inhibitory for Concanavalin A-pulsed normal volunteers' PMBC. The effect of the serum was not due to cytotoxicity, inadequate nutritional support or altered kinetics of DNA synthesis. High levels of IgM or IgG (both total and antiparasite) and high levels of triglycerides were found in patients' sera.

Cellular and humoral bases of hemorrhage-induced depression of lymphocyte function.

Bacterial infection often occurs after trauma and hemorrhage and is believed to be a reflection of a compromised host defense system. In the present study, the effect of hemorrhage on phytohemagglutinin-induced lymphocyte proliferation was investigated. Lymphocytes obtained from rats 2 h after blood withdrawal in an amount equivalent to 30% of total blood volume showed a 48% reduction in proliferative response as compared to cells obtained from the same animal before bleeding. This depression in lymphocyte proliferative capacity appeared to be due to a serum factor or factors induced by hemorrhage. The hemorrhage-induced serum factor(s) is heat-stable, dialyzable, and has an apparent molecular weight between 13,000 and 23,000 on gel filtration chromatography. The hemorrhage-induced factor seems to suppress lymphocyte proliferation in a rapid and irreversible manner. This abnormality in host defense mechanisms may contribute to the increased incidence of sepsis present after trauma and hemorrhage.

Effects of a Novel Compound (AL 721) on HTLV-III Infectivity in Vitro

This letter reports the results of a study indicating that AL 721 a lipid compound with a demonstrated ability to extract cholesterol from cellular membranes inhibits human T-lymphotropic virus type III (HTLV-III) infection of human peripheral-blood lymphocytes or an immortalized helper T-cell line (H9). When H9 cells were incubated with HTLV-III for 4 days reductions were detected in cell numbers and there were significant levels of reverse transciptase p15 and p24. After H9 cells were infected with HTLV-III in the presence of AL 721 there was a reduction in viral infectivity. The median dose for this effect was 100 mcg/ml of AL 721. It is not clear whether the virucidal effects of AL 721 are due to its actions on the retroviral envelope or on the host cell membrane. In preliminary clinical trials with elderly human subjects daily oral doses of AL 721 over a 6-week period restored diminished lymphocyte proliferative capacity and were well tolerated. AL 721 is considered a promising new candidate for investigation in the treatment of acquired immunodeficiency syndrome (AIDS) especially since it does not produce the side effects that are associated with reverse transcriptase inhibitors.