Involvement of phosphatidylinositol-3 kinase-Akt and nuclear factor kappa-B pathways in the effect of frutalin on human lymphocyte.

The mechanisms involved in the mitogenic effect of lectins are not fully understood and are thought to involve a cascade of intracellular signals related to T cell receptor activation. This study shows that frutalin, the alpha-D-galactose-binding lectin from Artocarpus incisa seeds, is a potent mitogenic activator of human lymphocytes. This effect is inhibited by D-galactose and PI3K inhibitors, and is accompanied by an increase in IL-2 receptor expression and by a PI3K-dependent IL-2 gene expression and IL-2 protein synthesis. Frutalin also induces Akt-phosphorylation and activates NF-kappaB, inducing its translocation from the cytosol to the nucleus. Both effects are blocked in the presence of D-galactose or by PI3K inhibitors. In summary, frutalin, interacting with alpha-D-galactose, activates signaling pathways related to TCR, and thereby triggers PI3K/Akt and NF-kappaB pathway, which modulates T cell proliferation, IL-2 synthesis and IL-2R expression. Frutalin might be a useful tool to study intracellular mechanisms following T cell activation that link upstream signaling pathways to downstream events.

Off-pump coronary artery bypass grafting does not reduce lymphocyte activation.

OBJECTIVE: In this study, we test the hypothesis that off-pump coronary bypass surgery might result in less lymphocyte activation than on-pump coronary surgery. We also study the behavior of lymphocyte activation markers during and after surgery. BACKGROUND: Coronary artery bypass surgery is known to be associated with changes of inflammatory mediators, immune function, and early phase lymphocyte activation, which could cause postoperative lymphopenia and lymphocyte unresponsiveness. METHODS: We studied lymphocyte activation response in 28 patients randomized to off-pump (n = 13) or on-pump (n = 15) coronary artery bypass surgery. Expression of CD25, CD26, CD69, and DR on T (CD3+) and B (CD19+) lymphocytes on peripheral blood was assessed through flow cytometry. RESULTS: The response of T lymphocytes and their activation markers, as well as B lymphocytes and their activation markers, was similar after on- and off-pump surgery. Overall, T lymphocytes decreased to the lowest level 9 h after surgery and tended to increase later. For B lymphocytes, there was early reduction with increase on the 1st postoperative day. There was early activation of CD69+ and late activation of CD25+ on T lymphocytes. For B lymphocytes, there was early activation of CD69+ and late activation of DR+. CONCLUSIONS: (1) Compared to on-pump cardiopulmonary bypass, off-pump surgery does not reduce lymphocyte activation. (2) Coronary bypass surgery causes the early activation of lymphocytes, as evidenced by the increased expression of lymphocyte activation markers.

Interleukin-2 inhibits proliferation of HPV-associated tumor cells and halts tumor growth in vivo.

Previous studies have shown inhibition of cervical cancer cell growth by treatment with high concentrations of IL-2. In the present study, we evaluated the in vitro and in vivo effects of recombinant human IL-2 on HPV-associated tumor cells (3T3-16). Treatment of 3T3-16 cells with rhIL-2 for 72 h inhibited cell growth in a dose-dependent manner and this effect was evidenced at nanomolar concentrations. These tumor cells expressed mRNA for beta and gamma subunits of the IL-2 receptor, which are required for signal transduction. In experiments to explore the effect of IL-2 on the growth of the HPV-associated tumor, mice received rhIL-2 through different routes: (i) intraperitoneal; (ii) subcutaneous, at the tumor inoculation site; or (iii) subcutaneous, distant from the tumor inoculation site. An effective antitumor response was observed only in those animals that received IL-2 at the tumor site (P<0.01). These results indicate the potential adequacy of therapeutic strategies based on local administration of rhIL-2 for cervical carcinoma, not only based on the ability of this cytokine to stimulate cellular-mediated immunity but also because of its direct effects on tumor cells.

Comparative and prospective study of different immune parameters in healthy subjects at risk for tuberculosis and in tuberculosis patients.

It has not been fully elucidated which of the components of the immune response against Mycobacterium tuberculosis is indicative of resistance or susceptibility. The aim of this study was to identify an immune parameter that could be indicative of either resistance or susceptibility to M. tuberculosis infection. We prospectively studied (three determinations, at months 0, 8, and 12) 15 patients with chronic pulmonary tuberculosis and 42 healthy individuals with a recent and frequent contact with tuberculosis patients. Peripheral blood mononuclear cells were stimulated with a whole-protein extract or the 30-kDa antigen of M. tuberculosis for 6 days, and several immune parameters were determined. No consistent differences between tuberculosis patients and healthy controls were detected in most immune parameters studied, including the expression of different activation antigens, cytokine secretion, lymphocyte proliferation, and nitric oxide production. However, the synthesis of tumor necrosis factor alpha, the intracellular detection of gamma interferon, and the apoptosis of monocytes under certain culture conditions tended to show clear-cut differences in cells from patients and controls (P < 0.05 in all cases for most determinations). Nevertheless, when results were analyzed on an individual basis, it was evident that a significant degree of overlapping of values from patients and controls occurred for all parameters studied. We conclude that although the immune parameters tested do not allow the identification of individuals susceptible to M. tuberculosis, the specificity and sensitivity of some of them could be improved through future studies.

Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite.

Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 microM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-alpha, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.

Brazilin augments cellular immunity in multiple low dose streptozotocin (MLD-STZ) induced type I diabetic mice.

Brazilin, an active principle of Caesalprenia sappan, was examined for its immunopotentiating effects in multiple low dose streptozotocin (MLD-STZ) induced type diabetic mice. Brazilin was intraperitoneally administered for 5 consecutive days to MLD-STZ induced type I diabetic mice. Delayed type hypersensitivity, Con A-induced proliferation of splenocytes and mixed lymphocyte reaction, which had been decreased in diabetic mice, were significantly recovered by the administration of brazilin. Brazilin increased IL-2 production without affecting suppressor cell activity. Con A-induced and IL-2-induced expression of high affinity IL-2 receptors were also enhanced by brazilin. These results indicate that brazilin augments cellular immune responses, which are suppressed in the MLD-STZ induced type I diabetic mice, by increasing IL-2 production and responsiveness of immune cells to IL-2.

Contribution of the expression of ICAM-1, HLA-DR and IL-2R to the diagnosis of acute rejection in renal allograft aspirative cytology.

Acute rejection is associated with a poor long-term prognosis for renal allografts. Sequential fine-needle aspiration cytology (FNAC) has been used to monitor rejection. However, FNAC diagnoses rejection only when the infiltrating cells are already damaging the graft and, in some borderline cases with a low increment of inflammatory cells in the graft, FNAC lacks the specificity to diagnose rejection. In these cases, the number of inflammatory cells within the graft can decline, stabilize or increase with time. In this study, we sought to determine whether the analysis of the expression of ICAM-I, HLA-DR and IL-2R along with borderline FNAC results increases the specificity to diagnose rejection. Of 117 FNAC samples taken from 24 patients after renal transplantation, 85 (72%) were considered suitable for cytological analysis. Of these patients, 9 (37%) did not suffer an acute cellular rejection (ACR) episode and 15 (63%) had at least one ACR episode. ICAM-1 and IL-2R were studied using an immune-peroxidase technique. The ICAM-1 results are expressed as the percentage of tubular cells in the aspirate stained with this marker and the IL-2R results are expressed as the absolute number of positively stained lymphocytes in the whole cytopreparation. With a total corrected increment (TCI) of > 3 there was a sharp increase in the specificity index for rejection that reached almost 100% at a TCI of > or = 4. Sensitivity for rejection at this level was only 20%. Between a TCI of 2.5 and 2.9 the sensitivity increased to 75%, with specificity for rejection around 75%. There was an upregulation of ICAM-1 and IL-2R when FNAC diagnosed rejection but with a large overlap of the results when compared either to normal graft or acute tubular neurosis. The mean TCI during the week preceding the rejection episode was 2.5 and the TCI reached a mean value of > or = 3 only during rejection. The peak ICAM-1 and IL-2R expression occurred during the week preceding the clinically evident rejection episode. The expression of ICAM-1 by > or = 70% of the tubular cells increased the specificity for rejection of a TCI of > or = 2.5 to 100%. In the same way, the specificity for rejection increased up to 90% when eight to ten IL-2R-positive lymphocytes were seen along with a TCI of > or = 2.5. There was no further increase in specificity after that. A specificity index of 100% for rejection could be obtained for moderate levels of both ICAM-1 (70% or more tubular cells) and IL-2R (eight or more lymphocytes). ICAM-1 expression in 70% or more tubular cells and/or IL-2R expression in eight or more lymphocytes was found in 58% of the FNAC aspirates with a TCI between 2.5 and 2.9. In conclusion, the expression of IL-2R in lymphoid cells and ICAM-1 in tubular cells was upregulated during rejection episodes and the upregulation preceded both the clinical and the routine FNAC diagnosis of rejection by 1 week. The ddition of these markers to the FNAC increased substantially the specificity of the FNAC to diagnose rejection.

Brazilin modulates immune function mainly by augmenting T cell activity in halothane administered mice.

Previously we reported that brazilin, the main principle of Caesalpinia sappan, was able to improve the altered immune functions caused by halothane administration in mice. To elucidate the mechanisms of its immunomodulating activities, the effects of brazilin on the functions of T cells and splenic cellularity were investigated. Brazilin decreased splenic cellularity and IL-2 production which had been augmented in mice treated with halothane (21.5% in olive oil, 10 mmol/kg) for 4 consecutive days whereas the reduced expression of IL-2 receptors by ConA or standard IL-2 was increased by brazilin treatment. These data indicate that halothane induced a dysfunction of T cells resulting in abnormal immune responses and these altered immune functions might be improved mainly by affecting the function of T cells.

Ouabain effects on activated lymphocytes: augmentation of CD25 expression on TPA-stimulated cells and of CD69 on PHA-and TPA-stimulated cells.

Ouabain (OUA) was capable of inhibiting peripheral blood lymphocyte (PBL) proliferation induced by phyothaemagglutinin (PHA) of phorbol ester (TPA), as measured by thymidine incorporation or cell cycle analysis. In this latter case it was possible to detect a block in the progression from G1 to S phase. This inhibition could not be reversed by interleukin (IL)-2 and was not due to an effect on CD 25 expression, as this molecule was only reduced in PHA cultures treated with OUA. Conversely, cultures activated by TPA and OUA showed an increased expression of CD25. The activation antigen CD69 was increased in both situation, suggesting that despite the absence of proliferative response the cells were being activated. The possibility that these cells were being deviated to the activation pathway leading to apoptosis is now under investigation. This study also suggested that CD25 induction may occur via different pathways, and that the selective effect of OUA for PHA-activated cells may become a useful tool for the understanding of the process.

Inhibition of human LAK-cell activity by the anti-depressant trifluoperazine.

The anti-depressive drug trifluoperazine (TFP) was studied on in vitro immune responses. TFP proved to be an inhibitor of lymphokine-activated killer (LAK) cells in its generative step, as well as in its effector phase. Natural killer (NK) activity and interleukin-2 (IL-2) or mitogen-induced lymphocyte proliferation were just as sensitive to the drug effects, whereas the division of tumor cells was more resistant. The mechanism through which TFP suppresses these lymphocytic systems remains unclear. It does not, however, affect an early stage of cellular activation as the addition of the drug as late as 24 h after the start of the culture was still inhibitory for lymphocyte mitogenesis. Neither the expression of CD25, nor that of CD56 was affected by TFP, and exogenous IL-2 was unable to overcome the suppression of proliferation. In relation to cell-mediated cytotoxicity, TFP partially interfered with the effector/target binding. However, addition of lectin to the assay did not overcome the inhibition of lysis produced by the drug. Although further work remains to be done, the effect of TFP on immune responses must be taken into consideration when treating immunosuppressed patients.

Longitudinal study on the production of and cellular response to interleukin-2 in patients with systemic lupus erythematosus.

Interleukin-2 (IL-2) has been proven to be a defective element in immune regulation in systemic lupus erythematosus (SLE). However, its course in time is unknown. We studied its production and cellular response in the peripheral blood cells of 30 SLE patients and 12 healthy subjects. In addition, we studied the spontaneous and lipopolysaccharide (LPS) induced production of IL-1, which have been found to be, respectively, increased and lowered in untreated SLE patients. Patients were studied at the outset, when still untreated, and at 1, 2, 6, 12, 18, and 24 months. At the outset, 18 had active disease and 12 were in remission. The decreased proliferative response of T cells to IL-2 and the deficient production of IL-1 upon LPS induction became normal after 6 months treatment, whereas the expression of high affinity IL-2 receptors took 18 months to become normal and the deficient production of IL-2 took 2 years. Despite clinical remission, the decreased capacity of T cells to absorb IL-2 persisted for 2 years. The effect of various prednisone dosages on the measured variables was evaluated. With intermediate doses of prednisone (20-45 mg), we observed the largest improvement in IL-2 production and in IL-1 production upon LPS stimulation. Higher doses of prednisone reduced also the spontaneous production of IL-1 and resulted in an increase in the expression of CD25+ cells. The addition of low doses of cytotoxic drugs (oral cyclophosphamide or azathioprine) resulted in an improvement in the capacity to absorb IL-2 and a reduction in spontaneous IL-1 production.(ABSTRACT TRUNCATED AT 250 WORDS)

E-selectin and interleukin-2 receptor alpha-chain expression in alopecia universalis.

A study of the histopathological abnormalities in a case of alopecia universalis was accompanied by immunohistochemical analysis of the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selectin (formerly known as endothelial leukocyte adhesion molecule-1) within the skin. ICAM-1 expression on follicular epithelium co-localized with intraepithelial mononuclear cells (MNC) positive for the interleukin-2 receptor alpha-chain (IL-2R) or HLA-DR. Aberrant expression of E-selectin was observed on dermal endothelium. Although restricted to one case, these new observations concerning the expression of E-selectin and IL-2R in alopecia universalis are consistent with the view that extravascular trafficking of MNC into follicular epithelium may play a key role in the pathogenesis of alopecia universalis and that use of agents that interfere with this process may be an effective therapeutic strategy.

Anti-CD3-activated T cells from chronic nonviremic HBV carriers are hyperreactive to monocytic accessory signals.

We analyzed T cell responses through the CD3 activation pathway in a group of chronic HBV carriers. PBMC stimulated with the mAb OKT3 showed higher proliferative response in HBV-DNA(-) carriers compared to HBV-DNA(+) carriers and to controls. In contrast, no differences in proliferative responses were observed between HBV-DNA(-) carriers and controls in cell cultures stimulated with immobilized 64.1 mAb (SPB-64.1) which induces proliferation in the absence of monocytes. We further examined T cell responses in the presence of monocytes and their soluble factors to immobilized OKT3 mAb (SPB-OKT3). Purified T cells did not proliferate to SPB-OKT3. When autologous monocytes were added, higher proliferative response, IL-2 production, and IL-2 receptor expression were observed in HBV-DNA(-) carriers than in controls. An enhanced cell proliferation was also obtained when monocyte supernatants were added to T cells cultured with SPB-OKT3. Moreover, when IL-6 alone or combined with IL-1 was added to SPB-OKT3-stimulated T cell cultures, a significantly higher increase in T cell proliferation was detected in HBV-DNA(-) carriers. Our results thus show a T cell hyperreactivity to accessory signals from monocytes (mainly IL-6) in HBV-DNA(-) carriers, that is probably related to an ongoing viral clearance.

Humoral and cellular immune response of adults from northeastern Brazil with chronic Trypanosoma cruzi infection: depressed cellular immune response to T. cruzi antigen among Chagas' disease patients with symptomatic versus indeterminate infection.

Infection with Trypanosoma cruzi can cause chronic Chagas' disease manifestations (cardiac, gastrointestinal), although most persons with chronic infection have no ill effects (indeterminate form). Cell-mediated immunity (CMI) responses are believed to be intrinsically important in the containment of T. cruzi and in the pathogenesis of Chagas' disease. Humoral and CMI responses were investigated in 70 T. cruzi-infected persons from an endemic area in northeastern Brazil and in 30 uninfected controls. An epidemiologic survey, physical examination, and blood evaluation were conducted for each subject. The 70 chronically infected persons were subclassified into three clinical groups: indeterminate, cardiac, and gastrointestinal. Serum was tested for antibodies to T. cruzi by hemagglutination assay, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay, and for autoantibodies to tubulin. Serum levels of soluble interleukin-2 receptor (sIL-2R), albumin, and C-reactive protein (CRP) were also measured to assess one parameter each of immunosuppression, nutritional status, and inflammation. The proliferative response of peripheral blood mononuclear cells (PBMC) to T. cruzi antigens, mitogen (phytohemagglutinin), and antigen-free controls was also assessed. Our data did not reveal any significant differences in serum levels of antibodies to T. cruzi, antibodies to tubulin, albumin, CRP, or sIL-2R among the subgroups of infected individuals. The data demonstrate differences in CMI responses. Trypanosoma cruzi trypomastigote lysate stimulated proliferation of PBMC from infected persons, but not uninfected controls. Patients with symptomatic Chagas' disease (cardiac and gastrointestinal groups) had decreased cellular responses to T. cruzi lysate (median proliferation index [PI] = 3), compared with those in the indeterminate group (median PI = 9; P < 0.005). Further investigations of the mechanism of this reduced CMI response in those with chronic disease may yield insights into the pathogenesis of Chagas' disease.

Cytoplasmic expression of a CD24-related epitope in human PHA activated normal T lymphocytes.

In this study we have analysed by immunoperoxidase (IPx) and indirect immunofluorescence (IIF) the intracellular and cell surface reactivity of VIB-E3 mAb, previously clustered as anti-CD24 antigen, on resting and activated normal human T lymphocytes. By IPx assay VIB-E3 mAb did not show reactivity with normal resting T cells. In contrast, the analysis of 11 different samples of PHA activated normal mononuclear cells, showed an intracytoplasmic expression of CD24. Kinetic studies showed that CD24 appears 24 to 48 h after PHA stimulation. To our knowledge, this is the first evidence that a CD24-related epitope is expressed in normal activated T lymphocytes.

Zidovudine therapy is associated with an increased capacity of phytohemagglutinin-stimulated cells to express interleukin-2 receptors. Pittsburgh AIDS Clinical Trial Unit.

Zidovudine therapy of AIDS patients has been shown to cause only transient improvements in the numbers of circulating CD4+ cells and the in vitro functional activities of cultured lymphocytes. The present studies were undertaken to determine whether prolonged zidovudine therapy enhanced reactivity in two sensitive assays of T-cell function: the ability of phytohemagglutinin (PHA)-stimulated cells to form T-cell colonies and their capacity to express receptors for the growth factor interleukin-2 (IL-2). Treated patients, studied over periods of 20-60 weeks, showed no improvement in colony formation at any time interval, even in plates supplemented with exogenous IL-2. However, mitogen-stimulated T lymphocytes showed a significant increase in the capacity to express IL-2 receptors (CD25). This enhanced expression resulted primarily from activation of the CD8+ cell subset.

Modulation of both interleukin 2 receptor expression and interleukin 2 production during experimental murine Trypanosoma cruzi infection.

A massive activation of T cells takes place during the early stages of a Trypanosoma cruzi infection in mice. We present data indicating that substantial amounts of interleukin 2 (IL-2) are secreted and IL-2 receptors are expressed during the period of increased proliferation (4-7 days post infection). Both concanavalin A-induced proliferation and IL-2 production are markedly decreased later in the acute infection (around 3 weeks post infection). This proliferation cannot be restored by externally added IL-2. Simultaneously, there is a drastic reduction in the number of both high- and low-affinity IL-2 receptors. The reduction is not attributable to the elimination of a particular T-cell population. In vivo administration of recombinant IL-2 failed to improve resistance to T. cruzi parasites as measured by parasitaemia and mortality.

Reduced suppressor cell response to Mycobacterium leprae in lepromatous leprosy.

We have previously shown that concanavalin A (ConA) induction of suppressor cell activity is impaired in patients with lepromatous leprosy (LL). In this study, we demonstrated that the proportion of cells bearing the Leu8 antigen (associated with suppressor-inducer cells) is low in LL patients and tends to normalize during the erythema nodosum leprosum (ENL) episode. Antigen-induced suppressor cell function was evaluated by a two-stage assay. In the first stage, peripheral blood mononuclear cells (PBMC) were cultured for 5 days either in the presence of gamma-irradiated Mycobacterium leprae or in tissue culture medium as a control. In the second stage, mitomycin C-treated suppressor or control cells were added to phytohemagglutinin (PHA)- or ConA-stimulated autologous PBMC. The results indicate that the ability of M. leprae to induce suppressor activity was lower in LL patients than in patients with tuberculoid (TT) and intermediate clinical (BB, BL, BT) forms and Mycobacterium bovis BCG-immunized normal controls. In ENL patients, the percent suppression was between that of TT and normal individuals. M. leprae-induced suppression was more effective on ConA- than on PHA-triggered T-cell proliferation in all groups. In contrast, normal PBMC cultured for 5 days in RPMI 1640 medium (N-C) and cells from patients with leprosy (TT-C and LL-C) had effects of their own on PHA- or ConA-induced proliferation. LL-C depressed the response to ConA and enhanced PHA-induced proliferation of autologous cells. Conversely, TT-C reduced PHA-induced proliferation and increased the ConA response. Suppression of proliferation could not be overcome with exogenous interleukin-2 and was not related to the induction of the Tac antigen. The abilities of LL, TT, ENL, and normal cells to proliferate upon PHA or ConA stimulus were similar, indicating that the defect in the generation of in vitro suppression by M. leprae in LL patients occurred during the induction period (step 1 of assay).

Immunoregulation in human schistosomiasis by idiotypic interactions and lymphokine-mediated mechanisms.

Anti-idiotypic (anti-Id) T cells from schistosomiasis patients or former patients proliferate upon exposure to polyclonal or monoclonal anti-soluble egg antigen (SEA) antibodies. Chloroquine does not inhibit, the response, which is induced by F(ab')2 (but not soluble Fab) fragments of these antibodies. Purified T cells from former patients require macrophages or exogenous IL-1 to respond to anti-SEA Ids and can respond to matrix-bound Fab fragments in the presence of IL-1. These anti-Id T cells recognize the Ids directly. Chronic schistosomiasis patients immunoregulate the production of a non-IL-2 lymphokine that stimulates IL-2 receptor expression on resting T cells. This regulation is reversed upon chemotherapeutic cure.