Effect of interleukin-2 and methylprednisolone on in vitro transformation of uremic lymphocytes

The functional relationship in vitro between mitogen-induced lymphocyte transformation, lymphocyte response to interleukin-2 (IL-2) and steroid, and production of IL-2 was examined in patients with chronic renal failure on hemodialysis (HD) or on continuous ambulatory peritoneal dialysis (CAPD). The lymphocyte responses to optimal stimulation with phytohemagglutinin, concanavalin A, and pokeweed mitogen were depressed in lymphocyte cultures from HD patients, while CAPD lymphocyte cultures responded normally. However, at suboptimal phytohemagglutinin stimulation both CAPD lymphocyte and HD lymphocyte responses were subnormal. Uremic lymphocyte cultures were more sensitive to the immunosuppressive effect of methylprednisolone. Addition of IL-2 normalized the phytohemagglutinin responses of suboptimally stimulated CAPD lymphocyte cultures and clearly improved the mitogen responses of the HD lymphocyte cultures. Furthermore, the increased uremic lymphocyte sensitivity to methylprednisolone was normalized by addition of IL-2 to the cultures. The measured IL-2 production had clearly decreased in the HD cultures after 48 h as compared to that of the control cultures. A similar but not significant trend was also seen in the CAPD cultures. Thus, it is suggested that a deficient production of IL-2 may partly explain the reduced lymphocyte response of uremic lymphocytes in vitro.     

Immunological studies in uremic and kidney transplanted patients

Some aspects of the cellular in vitro immune response were studied in uremic and kidney transplanted patients. In particular, the immunosuppressive effects of glucocorticoids were examined in uremic patients and in cadaver kidney graft recipients with special reference to clinical kidney transplantation. 1) In vitro, lymphocytes from patients on hemodialysis had impaired responses to stimulation with mitogen. Variations in the culture conditions including changes in; (i) mitogen concentrations, (ii) culture periods, (iii) aerobic growth conditions, (iiii) and cellular synthesis of prostaglandins did not normalize the uremic lymphocyte response. 2) A possible effect of hemodialysis per se could not be excluded. However, in short term experiments accumulation in uremic plasma of inhibitory factors and/or deprivation of supportive factors could only partly explain the decreased cell responses. 3) In vitro, glucocorticoids inhibit the proliferation of mitogen stimulated normal lymphocytes in a dose-dependent way. Moreover, the in vitro immunosuppressive effects of some glucocorticoids did not correlate with their anti-inflammatory potencies. In uremic cell cultures the in vitro lymphocyte sensitivity to glucocorticoids was 5-8 fold increased in comparison to the control cultures. 4) Lymphocytes from patients on peritoneal dialysis (CAPD) and non-dialyzed uremic patients had normal transformation responses but were also very sensitive in vitro to glucocorticoids. 5) Cytotoxic effector cell functions (NK and K) remained normal in hemodialyzed patients which is in contrast to the decreased transformation responses presupposing interleukin-2 (IL-2) dependent cell proliferation (DNA synthesis). Furthermore, both control and uremic NK and K cell functions were resistant in vitro to the suppressive effects of glucocorticoids. 6) The decreased mitogen response of patients on hemodialysis was improved by addition of IL-2 to the cell cultures. Moreover, IL-2 normalized the increased uremic sensitivity in vitro to glucocortiooids. In accordance, the production of IL-2 was decreased in mitogen stimulated cell cultures from patients on hemodialysis. There was no evidence that the impaired transformation responses of lymphocytes from patients on hemodialysis was due to a lack of cells positive for IL-2 receptors. These results suggested that a deficient production of IL-2 may be part of; (i) the decreased transformation response (ii) and the increased sensitivity in vitro to glucocorticoids of lymphocytes from hemodialyzed patients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of immune functions by human surfactant

Human peripheral blood mononuclear cells (MNC) were incubated in vitro with highly purified human surfactant to examine its effect on various T cell functions. Surfactant inhibited DNA synthesis by lymphocytes in response to concanavalin A (Con A), phytohemagglutinin (PHA), and in the autologous mixed lymphocyte reaction (AMLR). In contrast, surfactant had no effect on pokeweed-mitogen (PWM, T cell-dependent B lymphocyte mitogen)-induced DNA synthesis or on interleukin-2 (IL-2) receptor expression on T cells activated with PHA, Con A or PWM. Furthermore, surfactant had either no effect or enhanced (depending upon the concentration of IL-2 used) the response of exogenous recombinant IL-2 on IL-2-dependent T cell line, In vitro addition of recombinant IL-2 corrected the suppressive effect of surfactant on the AMLR. These data show immunosuppressive effect of surfactant on T lymphocyte functions.     

Lymphocyte stimulation and plasma inhibition in patients with malignant neoplasms. A comparative study with three mitogens.

The blastogenic response of lymphocytes from patients with malignant neoplasms was evaluated by stimulation with three phytomitogens (PHA, PWM, and Con A). The response of patient lymphocytes to all three mitogens was significantly lower than that of control lymphocytes, and most patients with abnormal PHA responses also responded abnormally to PWM and Con A. However, a few patients with normal PHA responses were abnormal to Con A, suggesting the suppression of a Con A-sensitive population. The observation that PWM responses were abnormal in patients with lowered PHA lymphocyte stimulation indicates that both T and B lymphocyte mitogen responses were suppressed in these patients. Plasma from patients was capable of either inhibiting or enhancing lymphocyte mitogen stimulation. However, inhibitory plasmas were generally from patients with abnormal mitogen responses.     

Cellular immunity in renal failure: depression of lymphocyte transformation by uraemia and methylprednisolone. Intra-individual consistency of lymphocyte responses to the in vitro suppressive effect of steroid

In vitro parameters of the cell-mediated immunity were assessed in patients with chronic renal failure on haemodialysis (HD). The in vitro mitogen responses of uraemic lymphocytes are depressed compared to control lymphocyte cultures. Prolonged or shortened incubation of uraemic cultures does not normalize the mitogen responses, and the difference is reflected in both DNA, RNA and protein synthesis of lymphocytes. Lymphocyte responses of control cultures incubated with uraemic plasma are similar to those of cultures with saline, and significantly stronger (p less than 0.05) than those of the uraemic lymphocyte cultures. The relative in vitro immunosuppressive effect of steroid is stronger in uraemic cultures. Thus the depressed uraemic lymphocyte responses may be associated with steroid-sensitive cellular interactions independent of incubation periods. However, both control and uraemic lymphocyte cultures have a reproducible individual in vitro lymphocyte response to the immunosuppressive effect of steroid.     

Changes in the expression of SRBC receptors on human lymphocytes caused by azathioprine and puromycine

The expression of human peripheral T lymphocyte receptors for sheep red blood cells in vitro was investigated. Changes in the expression of SRBC receptors were analysed in successive culture days depending on the stimulation of lymphocytes by PHA or Con A and the action of immunosuppressive agents, such as azathioprine and puromycine. The SRBC receptor variability of expression in cultures of whole peripheral blood lymphocytes was compared with that of T lymphocytes subpopulation isolated by the rosetting method. The experiments demonstrated: a) the appearance of different morphological E rosette types in cultures stimulated by plant mitogens, b) the inhibition of SRBC receptor expression by azathioprine and puromycine on lymphocytes from stimulated and non-stimulated cultures, c) higher sensitivity of SRBC receptors to immunosuppressive agents on cultured isolated T lymphocytes, d) the difference of azathioprine and puromycine action on the ability to form all types of E rosettes in stimulated and nonstimulated cultures.     

Kinetic Analysis of Interleukin 2 (IL-2) Production and Expression of IL-2 Receptors by Uraemic and Normal Lymphocytes

The in vitro immune response of lymphocytes from uraemic patients was studied by comparing the in vitro kinetics of interleukin 2 (IL-2) production, the mitogen-induced proliferative response, and the expression of IL-2 receptors by T lymphocytes. The IL-2 production in 26 uraemic cell cultures decreased significantly after 48 h of stimulation with mitogen compared with that of 24 control cultures. The lymphocyte responses to phytohaemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) increased linearly with time, but the responses of the uraemic cell cultures were significantly lower than those of the control cultures. The relative numbers of cells double-stained for both Tac (IL-2 receptor)/HLA-DR or Tac/Leu 2 were significantly increased in the uraemic cultures as compared with the control cultures at 48 and 72 h. A similar, but not significant, trend was also demonstrated for uraemic cells positive for Tac/Leu 3. These findings were also seen in uraemic lymphocyte cultures supplemented with exogenous IL-2. Thus, the IL-2 production of uraemic lymphocytes seems to be exhausted more rapidly than that of normal lymphocytes, and there is no evidence that the poor proliferative response of uraemic lymphocytes is due to a decreased relative number of cells positive for IL-2 receptors.     

Effects of cyclosporin A on the metabolism of unstimulated and mitogen-activated lymphocytes.

The immunosuppressive drug cyclosporin A (CS-A) reduces the magnitude of T-lymphocyte activation by all mitogenic lectins tested. However, in all cases a proportion of the activation observed is resistant even to very high concentrations of the drug. This proportion depends on the mitogen used, the responses to concanavalin A (Con A) and soybean agglutinin (SBA) being much more strongly inhibited than the responses to phytohaemagglutinin (PHA) or pokeweed mitogen (PWM). The differential effects of CS-A on lymphocyte activation by these mitogens could not be accounted for by the magnitude of the mitogenic response, the mitogen concentration used or the dependence of the responses on the presence of accessory cells, and they were maintained when several different procedures were used to assess the degree of activation. CS-A effectively inhibited inhibited lymphocyte activation. CS-A effectively inhibited lymphocyte activation only when added prior to, or very shortly after, the mitogen. Its ability to inhibit the response to PHA was lost more rapidly than that of Con A. The rate of protein synthesis by unstimulated lymphocytes was also affected by CS-A over the concentration range required to inhibit activation by mitogens. Although this effect was smaller than the inhibition of mitogen activation, it was highly significant and reproducible, and could not be accounted for by inhibition of spontaneous activation occurring in the unstimulated cultures.     

Mitogenic responses of canine peripheral blood lymphocytes to staphylococcal protein A

In order to produce long term lymphoid cell cultures from canine lymphocytes of known histocompatibility antigen specificities, mitogenic responses to staphylococcal protein A (SpA) were examined and compared with those of phytohaemagglutinin (PHA) and concanavalin A (Con A). SpA was found to be the strongest mitogen tested with significant responses to concentrations as low as 31 ng/ml. There was a decrease in responsiveness above optimal mitogen concentrations with SpA and PHA. Peak responses were observed at lower concentrations for longer incubation times. PHA showed a rapid fall off in thymidine uptake below optimal concentrations whereas the SpA dose-response curve was less steep and a shoulder or secondary peak of activity was observed at low SpA concentrations in some cases.Continuous SpA stimulation of lymphocyte cultures resulted in an initial period of cell proliferation followed usually by a second period of cell proliferation around week 7 of culture. To date, viable cell cultures have been maintained for up to 12 weeks in vitro.SpA lymphoblast cultures behave normally in microcytotoxicity tests for serologically defined DLA histocompatibility antigens and remain functional in natural killer (NK) and PHA induced cell mediated cytotoxic reactions against ⁵¹Cr-labelled tumour target cells but were not themselves susceptible as target cells for NK activity.     

Effects of Thymosin and Evidence of Monocyte Suppression of Both T- and B-Cell Functions in Two Cases of 'Common Variable Immunodeficiency'

We have studied two patients with common variable immunodeficiency (CVID) impaired cell-mediated immunity. and high percentages of monocytes in their peripheral blood. Removal of monocytes from cultures of peripheral blood mononuclear cells from both patients increased the in vilro responses to phytohaemagglutinin (PHA) and concanavalin A (Con A) but not to purified protein derivative (PPD), as measured by [³H]thymidine uptake. Similarly, supernatants of monocyte cultures from both patients. unlike supernatants of normal monocytes, suppressed the in vitro responses to PHA and Con A but enhanced the response to PPD by cultured mononuclear cells from the patients and from normal donors. Addition of unfractionaicd mononuclear cells from both patients 10 normal mononuclear cells suppressed both pokeweed mitogen (PWM) stimulation and IgG production: this effect was abrogated by removal of monocytes from the patients' mono-nuclear cell populations. The effect of thymosin on both patients' mononuclear cells was assayed in vitro. Thymosin was ineffective in vitro with cells from the first patient: for the other patient. [³H]thymidine uptake by mononuclear cells stimulated with PPD increased. whereas uptake by Con A-stimulated cells decreased, as did the percentage of E rosette-forming cells, providing further evidence of heterogeneity of the CVID syndrome. The effects of thymosin were also dependent on monocytes.     

Cutaneous tumor of northern pike, Esox lucius L. I. In vitro mitogenic responses of mononuclear cells from lymphoid tissues and tumor

To evaluate functional characteristics of lymphocytes from the northern pike, Esox lucius L., mononuclear cells were isolated by Ficoll-Isopaque gradients from lymphoid organs and cutaneous tumors of normal and tumor bearing pikes. The cells were tested in a lymphocyte proliferation assay in medium supplemented with fetal calf serum or autologous plasma using three concentrations of PHA, Con A, tuberculin PPD, and LPS. Lymphocytes of northern pike could be triggered to DNA synthesis in vitro. However, no clearcut anatomical partitioning of mitogen responses was found, since the mean optimal proliferation indices for each mitogen were similar in blood, head kidney, and spleen, while cells from head kidney, the equivalent of bone marrow, were stimulated as well with the murine T cell mitogens PHA and Con A as with the B cell mitogens PPD and LPS. Tumorous pikes seemed to have an apparently normal lymphocyte population since they responded by blastogenesis as well as normal pikes. Tumor cells exhibited a high basic metabolic rate and reactivity to mitogens was largely lacking.     

Immunological Studies in the Acquired Immunodeficiency Syndrome

The lymphocyte transformation responses to mitogens (phytohaemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM)), allogeneic cells, and the antigen-purified protein derivative (PPD) were studied in six acquired immunodeficiency syndrome (AIDS) patients and in six healthy controls, each of whom was HLA-DR- and mixed lymphocyte culture (MLC)-identical with one of the AIDS patients. No evidence of suppression was observed when irradiated or non-irradiated AIDS peripheral blood mononuclear cells (PBMC) were added to cultures of HLA-DR-identical PMBC from healthy controls stimulated with the strong mitogens PHA and Con A or with allogeneic cells, but suppression may be involved in the decreased responses in cultures stimulated with PWM or PPD. Addition of supernatants from macrocultures of AIDS cells did not suppress responses of control PBMC. Thus, suppression by any lymphocyte subset or soluble factor alone cannot explain the generally severely depressed transformation responses in AIDS. Addition of heavily irradiated HLA-DR-identical PBMC from healthy controls or supernatants from these cultures led to increased responses in cultures of mitogen-stimulated AIDS PBMC and in some cultures of antigen or allogeneic cell-stimulated AIDS PBMC, which were of the same magnitude as seen after the addition of commercially obtained T-cell growth factor (TCGF). This indicates that AIDS cells are deficient in producing TCGF. Heavily irradiated AIDS PBMC were capable of restoring the transformation responses to mitogens and antigens of purified HLA-DR-identical normal T cells, indicating that AIDS cells have a normal antigen-presenting capacity and interleukin (IL-1) production. However, AIDS PBMC had a very poor capacity to stimulate normal PBMC in MLC. Together, our experiments suggest that the immune deficiency in AIDS cells may be partially due to a decreased capability of T lymphocytes to produce TCGF and that a decreased number and/or function of dendritic cells may also be involved.     

Modulation of rat lymphocyte transformation by plasma fibronectin

Purified rat plasma fibronectin (Fn) was studied for its ability to influence nonspecific lymphocyte transformation to various mitogens. Fn added to lymph node cells (LNC) stimulated with phytohemagglutinin (PHA), concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) resulted in a noncytotoxic dose-dependent inhibition of the blastogenic response. Effective inhibition (greater than 50%) of LNC responses to PHA and LPS occurred with concentrations of Fn that ranged from 10-50 micrograms/culture. The proliferative response to Con A was less affected by the presence of Fn: 50% inhibition was observed only with the highest concentration of Fn studied. Fn at low concentrations was more effective than Fn-depleted plasma in inhibiting lymphocyte responses to PHA. Optimal expression of the regulatory activity of Fn occurs during and following the peak proliferative period for both PHA and LPS responses. In order to evoke maximum inhibition it is necessary for Fn to be present within 12 hours following stimulation of LNC with mitogen. Inhibition does not appear to be the result of Fn-mitogen complexes which reduce the amount of mitogen available for stimulation, since increasing concentrations of either PHA or LPS did not significantly reduce the inhibitory effect. Furthermore, inhibition was not influenced by the concentration of fetal calf serum present in the culture medium. Thus, Fn may function as an important nonspecific immunoregulatory factor at inflammatory sites and in areas of tissue repair.     

Lymphocyte proliferation in response to exercise

Lymphocyte proliferative responses are often used to evaluate the functional capacity of the immune system in response to exercise. Blood mononuclear cells (BMNC) are stimulated in vitro with polyclonal mitogens and the incorporation of ³H-thymidine into the DNA reflects cell proliferation. The BMNC are most often stimulated with either phytohaemagglutinin (PHA), poke weed mitogen (PWM), concanavalin A (Con-A), interleukin-2 (IL-2), or purified derivative of tuberculin (PPD). The literature concerning lymphocyte proliferation and exercise is reviewed with respect to the type and intensity of exercise, and also the effect of training status. The proliferative responses to exercise are highly heterogeneous, the most consistent finding being that PHA-stimulated cell responses decrease during exercise which may reflect a decreased fraction of CD3+ cells. In contrast, reduced, elevated or even unchanged lymphocyte proliferative response to PHA, PWM, Con-A, IL-2 and PPD have been demonstrated in the recovery period following exercise. Also variable responses are present in trained athletes compared to less fit subjects. Even though this may reflect that the time of ³H-thymidine incorporation into lymphocytes varies, we conclude that a functional evaluation of the immune system in response to exercise cannot be based solely upon measurements of lymphocyte proliferation.     

Synergistic effect of N-acetyl-D-glucosamine (NAG) on mitogenic, antigenic and allogeneic stimulation of normal human lymphocytes

The effect of N-acetyl-D-glucosamine (NAG) on in vitro stimulated human peripheral blood lymphocytes was investigated. NAG was added to lymphocyte cultures stimulated by the mitogen PHA, the antigen PPD or to one-way-stimulated mixed lymphocyte cultures from unrelated donors. The results indicate that NAG (1-2 micrograms/ml) in appropriate experimental conditions has an enhancing effect on DNA and protein synthesis induced by PHA, PPD and allogeneic cells. Addition of NAG (1-2 micrograms/ml) to unstimulated lymphocyte cultures had no effect on DNA or protein synthesis.     

Effects of concanavalin A, phytohemagglutinin, pokeweed mitogen, and lipopolysaccharide on the replication and immunoglobulin synthesis by canine peripheral blood lymphocytes in vitro

Canine peripheral blood lymphocytes (cPBLs) were used to investigate the mitogenic effects of Con A (concanavalin A), LPS (lipopolysaccharide), PHA (phytohemagglutinin), and PWM (pokeweed mitogen) in vitro by measuring tritium-labeled thymidine [( 3H]thymidine) incorporation and immunoglobulin (Ig) secretion. An ELISA specific for canine IgG and IgM showed that cPBLs secreted significantly more IgG than IgM in response to mitogen concentrations from 30,000 to 0.03 ng/10(5) cells. The optimal stimulating dose of mitogen for lymphocyte response measured by IgG secretion was over a much narrower range of concentration than was the [3H]thymidine incorporation measured response. At a concanavalin A dose where there was increased [3H]thymidine incorporation with a decrease in IgG secretion, it appeared that an active suppression of the IgG response was induced.     

Inhibitory effects of corticosteroids and histamine on human lymphocytes

Both corticosteroids and histamine inhibit mitogen-induced lymphocyte proliferation. Their mechanisms of inhibition were studied in lymphocytes exposed to methylprednisolone (MP) both in vivo and in vitro. Either in vivo MP treatment or in vitro histamine incubation depressed lymphocyte responsiveness to mitogen stimulation. The combination of both treatments depressed the proliferative responses to a further degree, representing a shift of baseline proliferation by in vivo MP treatment. For in vitro MP studies, normal lymphocytes cultured with varying concentrations of MP were significantly less stimulated by phytohemagglutinin (PHA) and concanavalin A (Con A). Addition of histamine (1 x 10(-3) M) as well as MP in vitro led to a further inhibition of proliferative responses of these cells in an additive pattern in cultures of all MP concentrations. Normal lymphocytes incubated in vitro with histamine, but not with MP, resulted in a rapid rise in intracellular cyclic adenosine monophosphate (cAMP). These findings suggest that corticosteroid and histamine act independently through different mechanisms.     

In vitro lymphocyte response to the phytomitogens in untreated and treated patients with Hodgkin's disease.

In vitro lymphocyte response to phytohemagglutinins (PHA), concavalin A (Con A), and pokeweed mitogen (PW) was evaluated in untreated and treated patients with Hodgkin's disease (HD). The responding capacity to PHA was depressed, though not constantly, in the untreated patients compared with the response to lymphocytes from normal individuals. The depression was more evident, at group level, when the cells were stimulated with suboptimal concentrations of PHA. Radiotherapy constantly induced a strong decrease or a complete loss of the responding capacity of the cells which persisted at low levels for many months. Some years after the initial course of treatment, the response was clearly depressed, but it was better in patients in remission than during relapse. Splenectomy did not affect the responding capacity of the cells. The depressive effect induced by chemotherapy was apparently less marked and persistent than that of radiation. Con A- and PW-induced lymphocyte transformation usually paralleled the PHA-induced response. The depressed response to PHA was not due to an inhibitory activity of HD serum. Washed HD lymphocytes in fetal calf serum were not stimulated better than in autologous plasma, nor were HD sera able to depress the response of normal lymphocytes to PHA, Con A, PW, and PPD. Supernatants from HD lymphocytes cultured for 24 h without any stimulant, and extracts of these cells were also unable to affect the response of normal lymphocytes to PHA.     

Leopard frog (Rana pipiens) spleen lymphocyte responses to plant lectins. Kinetics and carbohydrate inhibition

Mitogenic effects of the plant lectins phytohemagglutinin (PHA) and concanavalin A (Con A) on leopard frog (Rana pipiens) spleen lymphocytes in vitro were examined. At 22 ± 1°C, maximum stimulation in response to PHA and Con A occurs on day 4 with 1 pg PHA and 5–10 μg Con A. Lymphocyte stimulation can be inhibited by adding N-acetyl-D-galactosamine (NADG) to PHA cultures or α-methyl-D-mannopyranoside (αMM) to cultures containing Con A at time zero. Saccharide inhibition is concentration dependent: maximum inhibition is obtained with 75 mM NADG or αMM. Similarities between the kinetics of amphibian and mammalian mitogen stimulated lymphocytes and carbohydrate inhibition of lymphocyte activation suggest that certain biochemical characteristics of mitogen stimulation and cell surface receptors for mitogens have been phylogenetically conserved since amphibians first evolved during the Devonian period approximately 350 million years ago.     

Altered regulation of mitogen responsiveness by suppressor cells in multiple sclerosis.

Suppression of the lymphocyte response to concanavalin A (Con A), phytohaemagglutinin (PHA) and protein A from Staphylococcus aureus (SpA) by Con A-induced suppressor cells was measured in twenty-four patients with recently active-recovering multiple sclerosis (MS), twelve with inactive MS and twenty-three healthy controls. Patients with recently active disease displayed significantly greater suppression of the response to Con A. Suppression of the responses to PHA and SpA did not differ among the groups. Lymphocyte stimulation in cultures not showing suppression was similar in all three types of subjects. These results suggest a disturbance of lymphocyte regulation in patients with recently active-recovering MS and illustrate the potential usefulness of measuring the suppression of responsiveness to several mitogens.