Defective interleukin-2 production and interleukin-2 receptor expression in pulmonary tuberculosis.

The evolution of Mycobacterium tuberculosis as an intracellular pathogen has led to a complex relationship between it and its host, the human mononuclear phagocyte. The products of M. tuberculosis-specific T lymphocytes are essential for macrophage activation for intracellular mycobacterial killing. However, dysfunction cell-mediated immune response to infection with M. tuberculosis may contribute to progressive primary infection or reactivation of endogenous foci of mycobacteria. Th1 cells produce IL-2, which is essential for proper cellular immunity. The aim of this study was to identify the variation in IL-2 activity and soluble IL-2 receptor (IL-2 R) in peripheral blood lymphocyte in patients suffering with pulmonary tuberculosis. A significant decrease in IL-2 and IL-2 receptor level was observed in patients with pulmonary tuberculosis when compared to normal controls. Our results suggested that patients with pulmonary tuberculosis had a defect in IL-2 production. Better understanding of these interactions will allow the development of increasingly specific immune-based interventions for prevention and treatment of tuberculosis.

Expression of Recombinant Porcine Interleukin-2 and Application of Its Antibody to Immunoassays

Interleukin-2 plays an important role in T lymphocyte proliferation and immune response regulations. In this study, porcine IL-2 cDNA was cloned from peripheral blood mononuclear cells, and recombinant porcine IL-2 (rpIL-2) was expressed in Escherichia coli. The size of rpIL-2 without signal peptides was about 15 kDa when determined by SDS-PAGE and Western blotting analysis. Anti-rpIL-2 antibody was produced from mice immunized with the purified rpIL-2, and its specificity was examined by Western blotting and ELISA. In the Western blotting assay, anti-rpIL-2 and anti-recombinant human IL-2 (rhIL-2) antibodies specifically recognized rpIL-2 and rhIL-2, respectively. However, anti-rpIL-2 antibody did not recognize rhIL-2, and anti-rhIL-2 antibody also did not react with rpIL-2 in the same assay. In ELISA, anti-rpIL-2 antibody strongly interacted with both rpIL-2 and rhIL-2, and anti-rhIL-2 antibody also efficiently recognized both proteins. Taken together, the specificity of anti-rpIL-2 antibody for rpIL-2 was demonstrated by Western blotting and ELISA. It was also shown that ELISA is more efficient than Western blotting in determining the species cross-reactivity of anti-rpIL-2 antibody.

DNA-mediated immunization of mice with plasmid encoding HBs antigen

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.

Chronic hepatitis B virus carriers have low lymphoproliferative responses to HBsAg and reduced interleukin-2 synthesis.

BACKGROUND: Chronic carriers of hepatitis B virus (HBV) have impairment of lymphoproliferative responses. Recently HBV infection of peripheral blood mononuclear cells (PBMC) has been reported. The defect in the proliferative capacity of carrier PBMC has not been correlated to the presence of HBV in these cells. METHODS: PBMC of fourteen HBV carriers and 14 healthy individuals were stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM) or anti-CD3 for 3 days and with HBsAg and purified protein derivative (PPD) for 6 days. The supernatants of unstimulated and PHA-stimulated PBMC cultures were bioassayed for interleukin-2 (IL-2); the supernatants of unstimulated and lipopolysaccharide (LPS)-stimulated cultures were bioassayed for IL-1. DNA extracted from PBMC was hybridized with a 32P-labeled HBV probe to look for HBV DNA. RESULTS: HBV carriers' PBMC showed impaired responses to PHA, PWM and anti-CD3. No carrier demonstrated lymphoproliferative response to hepatitis B surface antigen (HBsAg). Seven of eight carriers with impaired HBsAg-specific proliferative responses who were tested for their response to an unrelated antigen showed a positive response to PPD. PBMC from HBV carriers produced similar amounts of IL-1 as normal PBMC on LPS stimulation; however, they produced significantly lower amounts of IL-2 as compared to normal PBMC under both spontaneous and PHA-stimulated conditions. HBV DNA was demonstrable in the PBMC of all fourteen carriers. CONCLUSIONS: The abnormal immune function found in chronic HBV carriers may be a consequence of replicative viral infection of the mononuclear cells.

Cell-mediated immunosuppression in mice by street rabies virus not restored by calcium ionophore or PMA.

It is known that rabies virus can suppress the host immune system. In this study we demonstrate a depression of cell-mediated immunity in mice, peripherally infected with Thai street rabies virus. The cell-mediated cytolysis of spleen cells from mice increased transiently on day 5 after infection and declined rapidly thereafter until death. The proliferation of spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin or concanavalin A, was significantly suppressed during the course of infection. There was also a marked suppression of IL-2 secretion in parallel with a decrease of the T-cell proliferative response to mitogen. The suppression of T-cell proliferation was not restored by treatment with a calcium ionophore (A 23187) or phorbol 12-myristate-13 acetate (PMA).

Mecanismos de ativaçäo do linfócito T e induçäo da leucemia linfoma T do adulto pelo HTLV I / Lymphocyte T activation and adult T lymphoma-leukemia induction by HTLV I

A infecçäo pelo vírus linfotrópico de célula T, tipo I (HTLV I), endêmica em algumas regiöes do mundo, ganha conotaçäo principalmente pelo fato de induzir a leucemia linfoma T do adulto e paraparesia espástica tropical/mielopatia associada ao HTLV I. O conhecimento da fisiopatologia da transformaçäo da célula T auxilia tanto a compreensäo das vias normais de ativaçäo/proliferaçäo do linfócito, como no mecanismo de aparecimento de doenças linfoproliferativas. As estratégias usadas pelo vírus para induzir à proliferaçäo celular afeta o ciclo celular em diferentes estágios e em diferentes vias de sinalizaçäo. Seräo analisadas as principais vias envolvidas nessa questäo e alguns mecanismos de açäo do vírus.

Interleucina-2 en pacientes con Síndromes Linfoproliferativos / Interleukin-2 in patients with lymphoproliferative syndromes

Se estudió la producción de interleucina-2 (IL-2) en las células mononucleares periféricas de controles normales y de pacientes con sídromes linfoproliferativos. Los valores normales de esta linfocina fueron: 31,74 ñ 18,25 µ/mL. Se encontró elevada en 3 pacientes con leucemia linfoide crónica de células B en estadio clínico III; disminuida en un paciente con linfoma no hodgkiniano y en otro con leucemia de células peludas; y ligeramente baja en pacientes con leucemia linfoide crónica en estadios clínicos O-II. No se encontró correlación entre los valores de IL-2 y la hemoglobina, el conteo global de leucocitos y la administración o no de tratamiento, pero se halló una correlación positiva con el conteo de linfocitos

Sustained leves of ascorbic acid are toxic and immunosuppressive for human T cells

Ascorbate (vitamin C) can protect from oxidative damage to DNA and lipids that may lead to aging, cancer, and other dysfunctions. However, we find that purified human T cells deteriorate if maintained in ascorbate in culture for 18 hrs. or more; viability and Il-2 synthesis are over 90 percent curtailed by scorbate at 50 micrograms/ml. T cell proliferation and adhesion are severely suppressed at 10-25 micrograms/ml. Dihydro-ascorbate was much less toxic or suppressive. The suppressive effect of ascorbate appears irreversible, since removal of ascorbate after 18 hrs. did not restore the mitogenic response. Although moderate dietary levels of ascorbate often reach 250-1000 mg or more daily and appear beneficial, our data caution against sustained megadoses of ascorbate for treatment of patients with AIDS and cancer

Defective lymphoproliferative responses & interleukin-2 production in chronic renal failure patients.

Cellular immune responses were evaluated in 12 early renal failure (ERF) patients who were not on maintenance haemodialysis, 43 end stage renal disease (ESRD) patients on haemodialysis (HD) and 25 healthy volunteers. Peripheral blood mononuclear cells (PBMC) of ERF and ESRD patients on HD had a significantly diminished lymphoproliferative responses to phytohaemagglutinin and a monoclonal antibody to the CD3 (anti-CD3) receptor on T-cells as compared to normals. The interleukin-2 (IL-2) production by the PBMC was also significantly reduced in renal failure patients as compared to normals. These data suggest that both IL-2 dependent and IL-2 independent T-cell functions are defective in renal failure patients.

T cell responses in Hodgkin's disease: frequency distribution of IL-2 producing cells and quantitation of IL-2 produced per cell.

T cell dysfunction in Hodgkin's disease (HD) is well documented. Since interleukin-2 (IL-2) plays a pivotal role in T cell proliferation, we have investigated frequency distribution of IL-2 producing phytohaemagglutinin (PHA)-stimulated lymphocytes from HD patients compared to that of healthy donors using two limiting dilution (LD) culture systems in which autologous peripheral blood lymphocytes (PBL) and Epstein Barr Virus transformed allogeneic B lymphoblastoid cell lines (EBV-LCL) have been used as feeders. The latter provided better conditions for IL-2 production by single cells, as evident from the enhanced frequencies obtained (For healthy donors: 1/67 +/- 1545.5 using EBV-LCL and 1/1123 +/- 1.7438 using autologous PBL as feeders). The data showed significantly reduced frequency of IL-2 producing cells as well as reduced quantity of IL-2 produced per cell in HD even after using/EBV-LCL as feeders, the amount of IL-2 produced per activated responder cell in HD patients being 0.825-1.3 pg/well (p < 0.001) as compared to 1.48-2.43 pg/well in healthy donors. Thus, the EBV-LCL feeders did provide better culture conditions for estimating frequencies of functional T cells. However these cell lines were unable to restore in vitro the abnormalities in functional properties of T cells in HD.

Cell mediated immune response in patients with Chagas. Correlation with the presence of Chagasic cardiomyopathy

We are reporting data from a series of studies undertaken to evaluate if there was any correlation between the cell mediated immune response and the presence of Chagasic cardiomyopathy. Infected patients without evidence of heart pathology (INF), infected patients with Chagasic cardiomyopathy (CDM) and healthy controls, were studied. We evaluated: a) the correlation of the immune response with the presence of parasitemia; b) the suppression induced by Trypanosoma cruzi antigens using a costimulation assay; c) their production of IL2 when stimulated with antigens and mitogen; and d) the response of variable proportion of T cells subpopulations to parasite antigens. The immune response of INF was lower when parasitemia could be demonstrated. The negative regulation of the cell mediated immune response was higher in INF. The production of IL2 was lower in patients than in controls. CDM patients showed a tendency to produce less IL2 than INF. The outline of the response of variable proportions of subsets of T cells had a high concordance in CDM and no concordance in INF. These results show that there are differences in the cell mediated immune response of INF and CDM. The high concordance of the response of variable proportions of T cells, from CDM, suggests that this test could have a prognostic value

The effect of serum from patients with acute myocardial infarction on in vitro lymphocyte reactivity. II. Inhibition of IL-2 production.

Culture supernatants from concanavalin-A (con-A)-activated peripheral blood lymphocytes from healthy controls grown in the presence of sera from 20 patients 24 hours and 1 week after acute myocardial infarction (AMI) were tested for their mitogenic activity and for the presence of interleukin-2 (IL-2). Binding of exogenous IL-2 to activated lymphocytes from 10 patients was also determined. In supernatants prepared in the presence of patients' as compared to control sera, a significantly decreased mitogenic activity and IL-2 content were found. The mitogenic activity and IL-2 content in culture supernatants prepared with patients' sera collected 24 hours after the AMI (AMI I) and one week thereafter (AMI II) were significantly suppressed, and the degree of suppression in the 24-hour sera was significantly higher than in those collected after one week. No significant differences were observed in the binding capacity to exogenous IL-2 of activated patients' and control lymphocytes. The possibility is that immunosuppressive factors in the patients' sera, including cortisol, may suppress the patients' immune response acting through inhibition of IL-2 production.

In vitro effect of interleukin-2 on proliferative responses of peripheral blood T cells from leprosy patients

Because of the important role played by interleukin-2(IL-2) in T cell growth and differentiation, we investigated the effect of exogenous IL-2 on the proliferative response of peripheral blood mononuclear cells(PBMCs) from 77 leprosy patients. The proliferative responses of PBMCs from lepromatous leprosy(LL) or borderline lepromatous leprosy(BL) patients to M. leprae were significantly lower(cpm 6,051 +/- 803 for LL type; 4,951 +/- 2,529 for BL type) than those from tuberculoid leprosy(TT) or borderline tuberculoid leprosy(BT) patients (28,853 +/- 28,916 for TT type; 15,884 +/- 334 for BT type). To investigate the effect of exogenous IL-2, purified IL-2 was added at the start of culture at 100 unit/ml. There was an apparent increase in 3H-thymidine incorporation of M. leprae-stimulated PBMCs(18,723 +/- 6,503) in the presence of IL-2 compared to the results without IL-2(6,051 +/- 803) in LL patients. Twenty nine out of 33 LL patients belonged to the responders to IL-2 and four patients were nonresponders. Therefore we conclude that the defective cell mediated immune response in LL patients may result from diminished production of IL-2, but we can not exclude the possibility of diminished expression of the IL-2 receptor. And we suggest that the immunologic heterogeneous response of an individual to M. leprae is important to the pathogenesis of clinical disease in the same LL patients.

Bovine interleukin 2: production kinetics in normal and in vivo antigen-primed cattle.

Conditions influencing production kinetics of bovine interleukin 2 (IL-2), viz. cell concentration, mitogen and its concentration, length of incubation, nutrient medium and in vivo antigen-priming were investigated. Peripheral blood mononuclear cells (PBL) of outbred cattle of different age groups showed considerable variation in their ability to secrete IL-2 which possibly reflects their immune competence. Of the cultures initiated with PBL, 5 x 10(6) cells/ml cultured in serum free Iscove's medium and stimulated with 5 micrograms Con A/ml for 24 hr produced maximal amount of IL-2 activity. In vivo antigen-priming of bovine lymphocytes with the live attenuated rinderpest virus revealed that IL-2 production was not affected by rinderpest virus but the in vivo antigen-priming possibly resulted in concomitant production of suppressor factor(s) which suppressed the already produced IL-2. The implications of this factor(s) in relation to regulation of immune responses in the disease process are discussed.

Independent and cooperative bheravior of THY1 and CD3:TcR T cell signalling pathways

Normal resting spleen T lymphocytes form mice were stimulated in vitro by monoclonal antibodies (mAbs) against either Thy1 or CD3:TcR surface protein molecules. Although both mAbs were mitogenic, anti-Thy1 activation generated 5 times more IL2 secretiom than anti-CD3 activation under similar conditions. Priduction of IL-like activity was comparable for both Thy1 and CD3-mediated activation. In addition, non-mitogenic doses of anti-CD3 and anti-Thy1 (0.16microng/ml and 0.0125% ascites, repectively) mAbs induced T cell activation when provided together. These reults indicate that Thy1 signalling cooperates with the CD3:TcR pathway to activate T cells. However, the pathway is also regulated independently since IL2 production is larger when stimulated by anti-Thy1 than anti-CD3mAbs

Effect of polyadenylic.polyuridylic acid on the proliferative responsiveness of mouse thymus and spleen cells

The effects of polyadenylic.polyuridylic acid [poly(A).poly(U)] on in vitro proliferations of thymus and spleen cells from C57BL/6 mice were investigated. Mice were injected intravenously with 30 micrograms of poly(A).poly(U) or placebo. Two days later, thymus, spleen and peritoneal cells from these mice were prepared and cultured in pooled or non-pooled conditions. Cell proliferations were assessed by the technique of incorporation of tritiated thymidine. It has been revealed that the in vitro proliferations of thymus and spleen cells as well as the productions of interleukin-1 by peritoneal adhering cells and interleukin-2 by spleen cells were significantly enhanced in the cultures of cells from poly(A).poly(U)-treated mice. These enhancing effects were observed only in the cultures of pooled cells from mice whose genetic homogeneity is suspected. Furthermore, thymus cells from poly(A).poly(U)-treated mice acted as strong responder cells but not as stimulators in one way mixed cultures. Thus, the enhanced cellular responsiveness may be mediated by the increased production of cytokines and antigen recognitions of thymus-derived cells following activations via the adjuvant effect of poly(A).poly(U).

Reduction of interleukin 1 and interleukin 2 production by patients with advanced cancer

The concentrations of interleukin 1 (IL-1) and interleukin 2 (IL-2) produced in the supernatants of peripheral blood mononuclear cell cultures from patients with advanced cancer were measured to identify some of the causes of the immunological impairment characteristic of malignant disease. Mononuclear cells obtained from 19 cancer patients were stimulated to produce IL-1 and IL-2 and compared with those of healthy controls. A severe reduction of both IL-1 and IL-2 activity was observed. There was no correlation between the lower number of OKT4+ cells observed in these patients and the levels of IL-2 production. The removal of monocytes did not bring IL-2 levels to normal. Impaired IL-2 production could not be restored to normal by addition of IL-1. These results suggest that exogenous IL-01 and IL-2 may be useful in cancer immunotherapy

Cell-mediated immunity in patients with invasive carcinoma of the cervix

Multiple in vitro immune parameters were investigated in thirty-four untreated patients with invasive carcinoma of the cervix and in twenty-five controls. The parameters measured were percentages and absolute counts of T and B cells, percentage of T cell subsets, lymphocyte response to phytohemagglutinin (PHA) and concanavalin A (Con A), natural killer (NK) activity, antibody-dependent cellular cytotoxicity (ADCC), and interleukin 2 (IL-2) activity. Patients with invasive cervical carcinoma, as compared with controls, showed a decrease in the percentage and count of T cells, a decrease in the percentage of helper-inducer (CD4+) T cells, decreased CD4+/CD8+ ratio, depressed lymphocyte response to PHA and Con A, and depressed NK and ADCC activities. There were no significant differences in these immune parameters between early and advanced tumor stages. The levels of total lymphocytes, monocytes, suppressor-effector (CD8+) T cells, and B cells were similar to those of the controls. IL-2 productivity in patients was lower than that in controls. In patients with invasive cervical carcinoma, a decrease in the percentage of CD4+ cells was associated with depressed PHA response and decreased IL-2 productivity was correlated with the reduced percentage of CD4+ cells and decreased NK activity. This study shows a significant defect in an important immune surveillance mechanism in patients with invasive carcinoma of the cervix and suggests that impaired IL-2 activity production may be related to quantitative and qualitative alterations in lymphocyte subpopulations which play a major role in immune surveillance against cervical cancer.