IgG production in ''autoimmune'' chronic active hepatitis. Effect of prednisolone on T and B lymphocyte function.

In vitro IgG production was measured using peripheral blood mononuclear cells from patients with autoimmune chronic active hepatitis (CAH) to determine whether the increased serum IgG levels were related to abnormalities of T or B lymphocyte function. A marked increase in spontaneous and pokeweed mitogen-induced proliferation of IgG producing cells was observed in 30 patients with untreated autoimmune CAH when compared with 25 normal subjects and 21 patients with autoimmune disease in whom a remission had been induced and maintained by prednisolone (P less than 0.01). Co-culture experiments clearly demonstrated that abnormalities of T lymphocyte function in untreated autoimmune CAH were responsible for the heightened IgG production in vitro. Pre-incubation of T lymphocytes from untreated patients with 5 X 10(-8)M prednisolone significantly reduced the number of cells producing IgG (P less than 0.05), suggesting that the modulation of the immune response following corticosteroid therapy is likely to be due to an alteration in T lymphocyte function.     

Abnormalities of B cell activation and immunoregulation in patients with chronic inflammatory liver diseases

The immunoglobulin production capacities of peripheral blood lymphocytes obtained from patients with various chronic inflammatory liver diseases and from normal individuals were compared. Using a reverse hemolytic plaque assay, immunoglobulin-secreting cells (ISC) were counted immediately after isolation (immediate ISC) and again after 6-day, in vitro cultivation without stimulant (spontaneous ISC) or in the presence of pokeweed mitogen, PWM (PWM-induced ISC). An increased number of immediate ISC were observed in patients with chronic active hepatitis (CAH) of the autoimmune type (n = 7) or with CAH type B (n = 32), probably reflecting a defect of the in vivo suppressor cell system as previously demonstrated. In vitro preincubation of cells with 5 x 10(-8) M prednisolone reduced the increase in the number of immediate ISC in patients with CAH of the autoimmune type. On the other hand, lymphocytes obtained from patients with CAH-type NANB (n = 9) and with primary biliary cirrhosis (PBC) (n = 12) showed an impaired capacity to generate ISC upon stimulation with PWM. Spontaneous ISC from patients' lymphocytes were not significantly different from those of normal individuals. Using allogeneic co-cultures with lymphocytes from normal individuals and from patients with CAH NANB hepatitis or primary biliary cirrhosis, we observed no increase in suppressor cell activity. Therefore, the diminished responses to PWM are probably attributable to an alteration in the peripheral helper T-cell compartment.     

Biological and molecular docking studies on coagulin-H: Human IL-2 novel natural inhibitor

Withanolide, coagulin-H (1), was evaluated for its effect on various cellular functions related to immune response including lymphocyte proliferation, and expression of interleukin-2 (IL-2) cytokine, and results were compared with prednisolone (2), a commonly used immune modulating drug. Coagulin-H (1) was found to have a powerful inhibitory effect on lymphocyte proliferation and Th-1 cytokine production. Inhibition of the phytohaemagglutinin (PHA)-activated T-cell proliferation by coagulin-H (1) was observed in a concentration dependent manner. A complete suppression of PHA-activated T-cell was observed at > or =2.5 microg/mL concentrations of compound (1) and this suppression activity was similar to that of prednisolone (2). Coagulin-H (1) also significantly inhibited IL-2 production by 80%. The interactions of coagulin-H (1) (a natural inhibitor) and prednisolone (2) (a drug) to IL-2 were also investigated in order to understand the differences in their effects on T-cell responses. This paper also describes the results of molecular docking study on IL-2 inhibition. Docking studies predicted that coagulin-H (1) binds to receptor binding site of IL-2 more effectively than prednisolone (2). Based on the computational and the experimental results, coagulin-H (1) was identified as a potential immunosuppressive candidate.     

Effect of (+)-cyanidanol-3 (Catergen) in chronic active hepatitis. (Catergen plus prednisolone versus prednisolone in a controlled study)

Thirty-six patients with biopsy-proved chronic active hepatitis (CAH) were randomized. Of these 18 patients, 15 of them HBsAg and/or anti-HBc positive, received (+)-cyanidanol-3 (Catergen, Zyma) in doses of 1.5 g daily for 6 months in addition to the previously started low dose (10-15 mg/day) prednisolone. The other 18 patients, 16 of them HBsAg and/or anti-HBc positive, continued on the former corticosteroid treatment exclusively. After 6 months an overall clinical-biochemical improvement was noted in 10 cyanidanol-treated and in 6 control patients, while the condition deteriorated in 4 drug-treated and 8 control subjects. Serum GPT activity decreased to less than twice the normal in 12/16 drug-treated and in 7/13 control subjects, gamma-GT fell to normal in 7/16 cyanidanol-treated and in 1/10 control patients, IgG fell to normal in 4/8 drug-treated and in 1/9 control patients who all had initially elevated values. The percentage of "active" E-rosette forming cells fell in both groups but the decrease was significant only in the control cases, i.e. cyanidanol inhibited the progressive fall in circulating T cells during the course of the disease. Lymphocyte proliferative response to phytohaemagglutinin stimulation, as measured by 3H-thymidine incorporation, significantly increased in the cyanidanol group. Anti-HBs titres were markedly raised in 7/15 drug-treated and in 2/16 control patients. Thus, cyanidanol had some slight beneficial effect on biochemical liver function tests and the immunological activity in CAH patients. Its use may be recommended as an adjuvant constituent of the complex therapy of the disease.     

T suppressor cell function and number in children with liver disease.

Con A stimulated suppressor cell function and the proportion of suppressor T cells were reduced in children with untreated chronic active hepatitis (CAH) but were normal in corticosteroid treated CAH patients, patients with severe acute hepatitis and inactive chronic liver disease. Adults with CAH also have defective suppressor function but a normal proportion of T suppressor cells. This difference may account for the observation that relapse after treatment withdrawal is less frequent in children than in adults.     

Interleukin 2 synthesis in the presence of steroids: a model of steroid resistance.

In this study interleukin 2 (IL-2) synthesis by human lymphocytes in the presence and absence of prednisolone in a group of normal subjects has been assessed. An association between suppression in vitro of induced phytohaemagglutinin-blastogenesis by prednisolone and synthesis of IL-2 was found. Those subjects whose lymphocytes are identified as steroid-resistant have significantly higher IL-2 activity in the supernatants of both steroid and non-steroid treated lymphocyte cultures than steroid sensitive subjects. The addition of exogenous IL-2 was found to ablate the suppressive effects of steroids on lymphocyte blastogenesis. These results suggest that significantly greater activity of IL-2 in the culture supernatants of steroid resistant subjects may represent a mechanism for glucocorticoid resistance in vitro and help explain the relationship between increased loss of grafts and steroid resistance in renal allograft recipients.     

Effect of Long-term Corticosteroid Therapy on Monocyte Chemotaxis in Man

The influence of prednisolone on monocyte chemotactic activity in vitro at prednisolone concentrations comparable with those achieved in man following oral dosage has been investigated. Chemptactic activity of monocytes from each of sixteen normal subjects was suppressed by concentrations of prednisolone as low as 25 ng/ml (suppression of chemotaxis, 20%). Maximal suppression occurred at 100 ng/ml (suppression of chemotaxis, 48%) and no significant increase in suppression was produced by increasing the concentration to 200 ng/ml (suppression of chemotaxis, 53%). In contrast, monocytes isolated from ten patients receiving corticosteroid therapy showed no significant suppression of chemotactic activity when exposed to these concentrations of prednisolone, even though they exhibited a normal ability to respond to a chemotactic stimulus. The lack of suppression of monocyte chemotaxis in patients receiving corticosteroid therapy is unexplained, but may represent a change in the circulating monocyte or lymphocyte populations.     

Lymphokine-induced suppressor B cells.

Accessory activity required for in vitro antibody production by murine B cells is mediated by macrophage- and helper T-cell-derived lymphokines (IL-1 and TRF, respectively). The synergistic helper activity that occurs when TRF is added early rather than late in the response cannot be attributed to the initiation. We found that the suboptimal helper activity which occurs when TRF is added early rather than late in the response cannot be attributed to the inactivation of the mediator, but results instead from the generation of helper factor-induced suppressor cells. Serological examination revealed that these suppressor cells exhibit the cell surface phenotype (Thy 1-, Ia+, IgG+, IgD+) of a B cell. In an attempt to attribute the generation of suppressor B cells to a distinct T-cell product we examined two T-cell mediators believed to be present in TRF, namely IL-2 and a second factor recently introduced as (DL) TRF. Our results point to a dichotomy of the induction pathways involved in the generation of antibody-forming cells and the activation of suppressor B cells, the former involving IL-1 and IL-2, and the latter involving IL-1 and (DL) TRF.     

Suppressor T-cell activity in chronic hepatitis B-virus infection: relationship with the presence of HBV-DNA in serum

Suppressor T-cell activity and allogeneic T-cell response to concanavalin A (ConA) were investigated in 46 patients chronically infected with hepatitis B virus (HBV). Thirty-eight patients had chronic active hepatitis, seven of whom were superinfected with Delta virus, and eight were healthy chronic HBV carriers. T-cell suppressor activity was in the normal range in healthy carriers and in patients negative for serum HBV-DNA, independent of the e antigen status. In contrast, the group of patients positive for HBV-DNA exhibited a significant reduction in suppressor activity. Longitudinal studies in patients who cleared serum HBV-DNA demonstrated that suppressor T-cell activity became normal thereafter. These results suggest a relationship between suppressor T-cell function and the stage of viral replication in individuals with chronic HBV infection.     

In vivo immunopotentiating activity of thymopentin in aging humans: modulation of IL-2 receptor expression

The effect of thymopentin treatment was investigated in immunocompromised elderly subjects. Thymopentin was able to increase IL-2 production and IL-2 receptor expression, as assessed on PHA-activated blasts by percentage of Tac-positive cells and response to exogenous IL-2. After treatment, an increased precursor frequency, estimated by limiting dilution analysis, of PHA-responding lymphocytes was observed in two out of six subjects tested. In vitro experiments with thymopentin show that the drug was able to enhance blastogenesis by PHA of elderly lymphocytes but not of adult cells. These results indicate that (a) the increased IL-2 synthesis/IL-2 receptor expression may be the crucial mechanism of the immunopotentiating activity of the drug in elderly subjects and (b) an increased intrinsic T-cell responsiveness seems to be responsible for this immunopotentiating activity, although an increase in the size of the responsive T-cell pool could not be excluded.     

Cell-mediated immunity and biological response modifiers in insulin-dependent diabetes mellitus complicated by end-stage renal disease

Previous studies have shown several immunoregulatory abnormalities in insulin-dependent diabetes mellitus (IDDM). In this report we compared peripheral blood mononuclear cells (PBMC) from patients with IDDM complicated by end-stage renal disease (ESRD) to those from normal subjects and from patients with ESRD of different etiologies for their: natural killer (NK) and antibody-dependent cell-mediated cytotoxic (ADCC) activities; modulation of NK and ADCC activities by biological response modifiers (BRM) including purified human lymphoblastoid interferon, human recombinant alpha-2 interferon, human gamma interferon and human recombinant interleukin 2; proliferative response of T and B lymphocytes to concanavalin A (Con A), phytohemagglutinin and pokeweed mitogen, and ability to produce T-cell growth factor (interleukin 2; IL-2). PBMC of diabetic patients demonstrated significantly lower NK activity than normal and ESRD subjects. Upon treatment with BRM, NK activity was augmented and achieved normal levels. ADCC activity was not different from that of normal controls and exhibited similar increases when stimulated by BRM. The proliferative responses to Con A, phytohemagglutinin and pokeweed mitogen as well as IL-2 production in response to Con A stimulation were significantly lower in the IDDM group. Our results indicated that NK cells from patients with IDDM can respond to IL-2 with enhanced cytotoxicity, and, because activation of resting T cells by mitogenic stimuli depends on the production of IL-2 as well as the appearance of a receptor for IL-2, our finding of low levels of in vitro IL-2 production by PBMC from patients with IDDM may explain the depressed NK activity and the observed poor response to T-cell mitogens.     

The effect of ultraviolet radiation-induced suppressor cells on T-cell activity.

The suppression of contact hypersensitivity (CHS) after a single exposure to ultraviolet (UV) radiation provides an excellent model system with which to study both the activation and the mode of action of suppressor T cells. Suppression of CHS after UV radiation is mediated by hapten-specific suppressor T cells (UVTs). These cells have a broad range of activity: CHS and antibody production in vivo and the generation of cytolytic T lymphocytes (CTL) and T-cell proliferative responses in vitro are suppressed by UVTs. The present study is concerned with determining the target of UVTs. The UVTs could suppress the response to hapten-modified T-dependent antigens, such as trinitrophenyl (TNP)-modified sheep erythrocytes (TNP-SRBC) or TNP-conjugated bovine serum albumin (TNP-BSA), but had no suppressive effect on the response to a T-independent antigen, TNP-conjugated lipopolysaccharide (TNP-LPS). The UVTs also suppressed the generation of interleukin-2 (IL-2) in vitro. The suppression of CTL generation in vitro and CHS in vivo could be overcome by the addition of exogenous IL-2. These data suggest that UVTs suppress the immune response by affecting T-helper cell function.     

Glucocorticoids Enhance the in Vitro Ig Synthesis of Pokeweed Mitogen-Stimulated Human B Cells by Inhibiting the Suppressive Effect of T8+ T Cells

The in vitro effects of three potent glucocorticoids (GC) (dexamethasone, prednisolone, cortisone) on human lymphocyte functions were investigated. In pharmacological concentrations GC strongly suppressed lymphocyte transformation induced by T-cell mitogen (concanavalin A and phytohaemagglutinin). After pokeweed mitogen (PWM) stimulation GC enhanced the number of immunoglobulin (Ig)-producing cells without affecting the proliferative response. Mineralocorticoid aldosterone showed no effect. Addition of 3 X 10(-8)-3 X 10(-6) mol/l of different GC to PWM cultures significantly increased the number of Ig-secreting cells, measured by the plaque-forming cell assay. Experiments conducted with fractionated defined lymphocyte subpopulations showed that the T8+, a radiosensitive T suppressor cell, is more sensitive than the T4+ T helper cell to GC effects. It is concluded that GC in pharmacological concentrations display a dual effect on human lymphocyte functions in vitro: an inhibition of lectin-induced T lymphocyte proliferation and a rather selective inhibition of T suppressor cell function which leads to an enhanced B-cell maturation and Ig synthesis in PWM-stimulated cultures. No measurable direct effect on the B lymphocytes was noticed.     

In vitro immunomodulatory effects of interleukin-2 and thymosin fraction V in acquired immune deficiency syndrome

A monoclonal antibody (anti-Tac) that appears to bind the receptor for interleukin-2 (IL-2) was used to quantitate lymphocytes that express IL-2 receptors (IL-2R) in response to phytohemagglutinin (PHA) stimulation. 62 +/- 4% of the cells expressed IL-2R in response to PHA in twelve normal subjects compared to 22 +/- 4% in fourteen patients with acquired immune deficiency syndrome (AIDS) (P less than 0.001) and 50 +/- 6% in six patients with AIDS-related complex (P less than 0.1). There was no effect on IL-2R expression, when lymphocytes from seven controls were incubated with IL-2 (20 mu/ml) or thymosin fraction V (10 mu/ml) for 72 h. However, when the lymphocytes from seven patients with AIDS were incubated with IL-2, the IL-2R rose from 18 +/- 3% to 31 +/- 3% (P less than 0.005) and with a fraction V to 29 +/- 3% (P less than 0.001). In addition, IL-2 augmented the PHA-induced proliferative responses in patients with AIDS-related complex and AIDS and normal controls, whereas thymosin fraction V had no significant effect. Thymosin fraction V also enhanced the IL-2 production of PHA-stimulated mononuclear cells obtained from six patients with AIDS and six normal controls. These results suggest that both IL-2 and thymosin fraction V can modulate in vitro T-cell function in patients with AIDS.     

Association of decreased T-cell-mediated natural cytotoxicity and interferon production in Down's syndrome

Total peripheral blood lymphocytes (PBL) and isolated subpopulations from children with Down's Syndrome (DS) and age-matched healthy controls were investigated for their (1) natural killer (NK) and antibody-dependent cellular cytotoxic activities, (2) interleukin 2 (IL-2)-induced augmentation of NK activity, (3) lectin-dependent cellular cytotoxicity (LDCC), (4) ability of serum- and culture-derived soluble suppressor factor(s) to inhibit NK activity of normal lymphocytes, and (5) capacity to produce interferon (IFN) against tumor targets in vitro. T lymphocytes from DS patients demonstrated significantly decreased NK activity against K562 target cells compared to controls. DS lymphocytes also demonstrated a significant reduction in LDCC activity and IL-2-induced enhancement of NK activity. Furthermore, the ability of DS lymphocytes to produce IFN in vitro against K562 target cells was also significantly lower than that for normal PBL. Although sera from DS patients showed a significantly greater inhibitory effect on the NK activity of allogeneic normal PBL than normal sera, culture supernates from DS lymphocytes demonstrated suppressive effects comparable to culture supernates from normal PBL. These studies suggest an association between the decreased NK activity of T-cell subpopulations and lower IFN production by PBL from patients with DS.     

Correlation between the results of glucocorticoid therapy and in vitro effect of glucocorticoids on monocytes in asthma

The effects of glucocorticoids on monocyte morphology and function in vitro and the results of high-dose budesonide therapy in patients with non-severe bronchial asthma were analyzed. Before therapy with inhalation glucocorticosteroid (budesonide) characteristics of blood monocytes and the effects of different concentrations of prednisolone on these cells were studied in vitro by luminol-dependent chemiluminescence and computer-assisted phase-interference microscopy. High sensitivity of patients to budesonide was associated with pronounced in vitro inhibitory effect of prednisolone on monocyte activity, which was not observed in cases with delayed effects of therapy. Pronounced inhibitory effects of glucocorticoids on monocytes in vitro were observed in patients both resistant and highly sensitive to glucocorticoid therapy. Hence, the resistance of patients with non-severe asthma to high-dose budesonide therapy is not related to the weakening of the inhibitory effect of glucocorticoids on monocyte activity.     

Stimulation of splenic T-lymphocyte function by endogenous serotonin and by low-dose exogenous serotonin.

The modulatory effects of endogenous serotonin on splenic T-cell activity were investigated using two distinct approaches. The first approach showed that pretreatment of mice with p-cholorphenylalanine (PCPA) to deplete intracellular stores of serotonin reduced the capacity of their splenic T cells to proliferate and to express interleukin-2 receptor (IL-2R) in response to concanavalin A (Con A). These responses could be restored by the addition of serotonin to the spleen cell cultures. In contrast, PCPA treatment did not effect stimulation of spleen cells to produce IL-2. The second approach showed that T-cell proliferation to Con A as well as to IL-2 was diminished by the presence of antagonists to the serotonin-2 receptor (5-HT2R). The effects of low doses (100 ng/ml) of exogenously added serotonin on functions of normal spleen cells were also examined. At this low dose, serotonin stimulated splenic T-cell proliferation in response to IL-2, and enhanced both proliferation and IL-2 production in response to a suboptimal concentration of Con A. These results show autologous serotonin to be required for T-cell activation and that the activation of suboptimally stimulated T cells can be augmented with low doses of exogenously added serotonin. These data also suggest that the positive regulation of T-cell function by serotonin is mediated through 5-HT2R.     

Suppressor cell activity in viral and non-viral chronic active hepatitis.

Suppressor cell function was studied in twenty-nine patients with chronic active hepatitis (CAH) in relation to possible aetiological causes and activity of liver disease. All fifteen patients with evidence of viral aetiology (ten HBsAg-positive CAH and five non-A, non-B CAH) showed normal suppressor cell function independently of severity of liver damage. In contrast, fourteen patients with HBsAg-negative CAH, including four cases with circulating antibodies to the hepatitis B virus, demonstrated a significant reduction in supprpessor cell activity compared to control subjects. No significant difference was found in this group between cases with and without circulating autoantibodies. In four out of five HBsAg-negative patients tested serially suppressor cell defect correlated with disease activity suggesting an abnormality in the regulation rather than a depletion of suppressor cells. These results suggest that different mechanisms are responsible for autoimmunity to the liver in virus and non-virus-related CAH.     

T-Cell Abnormalities in Antibody Deficiency Syndromes

We studied two patients with hypogammaglobulinaemia, three with selective IgA deficiency, and four with selective extreme IgM deficiency for T-cell abnormalities in the circulating blood. Most patients had altered CD4+ helper/inducer and CD8+ suppressor/cytotoxic T-cell subsets, and an elevated percentage of HLA-DR+ and interleukin 2 (IL-2) receptor+ antigens on lymphocytes. One patient with the selective IgA deficiency had an absence of CD4+ T cells in blood. The lymphocytes of eight patients showed a diminished proliferative response and deficient IL-2 production in response to stimulation with phytohaemagglutinin or concanavalin A. The three patients with selective IgM deficiency were found to have a defective T-cell abnormality in IgM synthesis by B cells. It is interesting that CD8+ T cells of the two patients with IgM deficiency demonstrated suppressor cell function for IgM synthesis by the normal B and T cells.     

Aberrations of suppressor T cells in human graft-versus-host disease.

To determine whether imbalances in immunoregulatory T-cell subsets exist in patients with graft-versus-host disease, we analyzed T cells in three patients with acute and in six patients with chronic graft-versus-host disease after bone-marrow transplantation. The normal human peripheral-blood T-cell compartment is composed of 80 per cent TH2-and 20 per cent TH2+ T cells, and defined by reactivity with subset-specific heteroantiserums. Human suppressor cells are TH2+, whereas helper cells are TH2-. Patients with acute and chronic graft-versus-host disease had abnormalities in these populations, and their T cells frequently bore la-like antigens. Patients with acute disease lacked TH2+ cells, and the reappearance of this subset preceded the cessation of disease activity. Chronic disease, in contrast, was more heterogeneous. Suppressor cells were lacking in two patients but increased in the other four. Two of these four patients had TH2+, la+ T cells, suggesting in vivo activation of suppressor cells. Studies showing that these TH2+, la+ cells actively suppressed the in vitro immune response support this hypothesis and suggest that the immunoregulatory cells may profoundly affect the overall immune response.