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.     

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.     

In vitro immune functions in patients with minor, moderate, and severe kidney impairment

We examined the in vitro immune response of lymphocytes from 86 non-dialyzed patients with progressive renal failure and 48 healthy control subjects by comparing the production of interleukin-2, the mitogen-induced proliferative response and sensitivity to glucocorticoid of lymphocyte cultures. The patients were divided in three groups with minor, moderate, and severe uremia. The uremic lymphocyte responses to stimulation with concanavalin A (Con A) were significantly lower than those of control lymphocyte cultures. A similar but not significant decrease was also seen in phytohemagglutinin (PHA) and pokeweed mitogen stimulated cultures. There was no trend towards diminishing mitogen responses with decreasing renal function. The median interleukin-2 activity in the uremic cell cultures was decreased by 50% during the culture period but the decrease was not significant. However, PHA and Con A stimulated lymphocyte cultures from all groups of patients were significantly more sensitive to the immunosuppressive effect of methylprednisolone. Thus an increased sensitivity to the immunosuppressive effect of glucocorticoid can be demonstrated in vitro at an early stage of progressive renal disease.     

In vitro analysis of B lymphocyte function in uraemia.

We have investigated the immune responses in vitro of uraemic patients undergoing regular haemodialysis or continuous ambulatory peritoneal dialysis. Twenty-five healthy subjects were also studied as controls. In uraemic patients, the number of T and B lymphocytes were within the normal range, but proliferative responses to phytohaemagglutinin (PHA) were impaired. Spontaneous immunoglobulin plaque forming cell (PFC) responses by peripheral blood mononuclear cells (PBMC) from uraemic patients were significantly lower than those of healthy subjects. The PFC response of uraemic PBMC to the T cell independent polyclonal B cell activator (PBA) Epstein-Barr virus (EBV) was comparable to the response of the healthy subjects, indicating that uraemic B cells are still capable of synthesizing immunoglobulin. Pokeweed mitogen (PWM) induced PFC responses of uraemic PBMC were also normal, whereas the response to another T cell dependent B cell activator, Staphylococcus aureus Cowan I (SAC), was very low. Addition of indomethacin to PWM- and SAC-activated cultures of uraemic PBMC enhanced the PFC response to SAC, but had little effect on the PWM response. As full differentiation of B cells in response to SAC depends on helper T cells, we conclude that a defect in T lymphocyte function accounts for the reduced spontaneous and SAC induced production of immunoglobulin by uraemic PBMC. This defect may be mediated by an indomethacin-sensitive mechanism.     

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.     

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)     

Lymphocyte transformation in human plasma without addition of synthetic medium. A study of immune function in patients with chronic uraemia or diabetes mellitus.

The in vitro response of lymphocytes obtained from normal subjects, uraemic patients on haemodialysis and diabetic patients was studied using cultures containing either medium plus plasma (medium cultures) or plasma alone (plasma cultures). The study demonstrated that plasma alone can adequately support lymphocyte transformation induced by nonspecific mitogens (PHA and PWM) and allogeneic lymphocytes in mixed lymphocyte culture (MLC) reaction. This investigation further confirms our previously reported findings that uraemic patients undergoing haemodialysis have a normal lymphocyte response in MLC and to PHA and PWM. Plasma cultures give results similar to conventional medium cultures in subjects where lymphocyte transformation is normal. The lymphocyte hyporactivity observed in diabetics is, however, better shown in the plasma cultures. The suppressed response of the diabetic patient's lymphocytes to PHA and PWM both in the presence of autologous and normal AB plasma suggests intrinsic lymphocyte dysfunction as the explanation for impaired immune function. Plasma cultures may provide a better in vitro system for the evaluation of immune function in certain groups of patients where it is desirable to distinguish between intrinsic abnormalities of lymphocyte function and the effect of humoral immunosuppressive factors.     

Effects of autologous plasma on lymphocyte transformation in malaria and in acute protein-energy malnutrition. Comparison of purified lymphocyte and whole blood cultures.

Phytohaemagglutinin (PHA) induced lymphocyte transformation in whole blood and in purified lymphocyte cultures was investigated in Gambian children with acute Plasmodium falciparum malaria or with acute protein-energy malnutrition (PEM). Responses of purified lymphocytes cultured in the absence of autologous plasma were normal, with one exception. Autologous plasma depressed the response of purified lymphocytes to a low dose of PHA in several malaria and PEM patients. In whole blood cultures of 1 day and of 3 day duration, responses of several children with malaria or PEM were less than those of control children. Responses were not related to absolute lymphocyte counts. In 3 day, but not 1 day, cultures from control and malarious children, responses were inversely proportional to neutrophil counts. Cultures of whole blood and of purified lymphocytes in autologous plasma gave comparable results in 58 of 70 patients.     

Modification of lymphocyte responsiveness in vitro by lambda carrageenan compared with colloidal silica and depletion of surface adherent cells.

The effects of the immunosuppressive sulphated polygalactan lambda carrageenan on in vitro models of allograft immunity were compared with the effects of removing macrophages (surface adherent and/or phagocytic cells) by established methods. Carrageenan depressed the primary mixed lymphocyte reactions, but not to the same extent as the removal of macrophages. 2-Mercaptoethanol restored the response. Secondary mixed lymphocyte reactions and responses to phytohaemaglutinin were depressed by carrageenan but not by the removal of macrophages, and in these systems 2-mercaptoethanol failed to restore the responses of carrageenan-treated cultures. In contrast, cell-mediated cytolysis by presensitized lymphocytes was not affected by carrageenan or by colloidal silica. Carrageenan depressed cell-mediated cytolysis only if it was present during the sensitization of the effector cells. We conclude that carrageenan can have two dose-related effects in vitro: one on the macrophage and one on the responding lymphocyte.     

Mitogen-induced lymphocyte transformation in four different serum-free media

The proliferative responses of lymphocytes induced by the mitogens phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (ConA) were tested in four different serum-free lymphocyte culture media in which serum was replaced by commercially produced serum substitutes. Two of the serum-free lymphocytes cultures (SF-X and FEB-100) achieved proliferative responses to PHA and PWM comparable with those obtained in medium containing fetal calf serum, but only when the cell number was increased, whereas none of the lymphocyte cultures in serum-free medium showed adequate proliferative responses to ConA stimulation. The essential factors in serum-free media for optimal growth and mitogen stimulation of lymphocytes include insulin, transferrin, electrolytes, glutamine and a high cell concentration.     

Host immune status in uraemia. II. Serum factors and lymphocyte transformation.

A model of experimentally induced uraemia has been used to study the effect of serum from uraemic rats on the immune responsiveness of thymus-derived (T) lymphocytes. Splenic lymphocytes from normal or uraemic animals responded to mitogenic stimulation with concanavalin A to a similar degree when cultured in a tissue culture medium containing the maximum non-toxic concentration of normal or uraemic serum in the culture system (3%). Serum from uraemic animals, however, had an immunosuppressive effect if the serum was first dialysed for 24 hr before being added to the tissue culture medium. When an alternative vessel was used which allowed the concentration of serum in the medium to be increased to 10%, serum from severely uraemic animals markedly suppressed the capacity of lymphocytes from normal animals to respond to Con A. Thus while serum from uraemic animals can be shown to be immunosuppressive, the results of the experiments are influenced by the conditions in vitro. The type of culture vessel and the concentration of serum in the culture medium are particularly critical determinants. It is likely that variations in laboratory procedures have contributed to the differences of opinion on the effect of serum from uraemic individuals on lymphocyte function.     

Regulation of lymphocyte proliferation after influenza virus infection of human mononuclear leukocytes

Previous studies demonstrated that in vitro infection with influenza A viruses altered several functions of human monocytes-macrophages but did not detectably alter functions of human lymphocytes. However, both types of cells were infected, as determined by production and surface expression of viral antigens. In the current studies, human mononuclear leukocytes were infected in vitro and assayed for both influenza virus-induced proliferation and mitogen (phytohemagglutinin [PHAl)-induced proliferation, as well as for ability to stimulate proliferative responses by normal autologous leukocytes. The leukocytes showed proliferation in response to the infectious virus, but concomitant depressed proliferative responses to PHA. Coculture experiments suggested suppression of PHA-induced responses by the virus-infected cells. However, upon coculture with fresh autologous leukocytes (without PHA stimulation), both virus-infected macrophages and virus-infected lymphocytes induced autologous lymphocyte proliferative responses. Altered proliferative responses to mitogen stimulation after exposure to the virus were not due to diminished interleukin-1 production or diminished expression of HLA-DR by monocytes-macrophages. The expression of influenza virus antigens and resulting induction of autologous proliferative responses, combined with depressed mitogen-induced proliferation, may be important in human antiviral defense.     

Immunosuppressive effects of lymphocyte (type II) and leucocyte (type I) interferon on primary antibody responses in vivo and in vitro.

We have tested the hypothesis that type II interferon (IF), released by immune lymphocytes after in vivo stimulation with tuberculin, has immunosuppressive effects. Mycobacterium bovis (BCG) infected mice injected with tuberculin showed a very intense suppression of antibody response to sheep erythrocytes. Sera containing lymphocyte IF strongly inhibited primary immune responses to sheep erythrocytes in cultures. Addition of macrophages could not counteract the in vitro immunosuppressive effects of lymphocyte IF, suggesting that the main effect is exerted directly on lymphocytes. Sendai virus-induced leucocyte (type I) IF was also shown to have suppressive effects in vivo and in vitro. However, lymphocyte IF was shown to be much more immunosuppressive than a preparation of type I interferon with equivalent antiviral potency. Thus type II IF, as a product of activated lymphocytes, may have a major immunoregulatory role.     

Glucocorticoid inhibitory action on the response to PHA of residual circulatory lymphocytes in renal transplant patients following immunosuppressive therapy

The inhibitory effect of glucocorticoids on the in vitro response to phytohaemagglutinin of the residual circulating T lymphocytes in renal transplant patients during maintenance immunosuppressive therapy with glucocorticosteroids and azathioprine has been investigated. As in normal subjects, the steroid-induced inhibition of transplanted patients' lymphocyte response was inversely correlated with the mitogen concentration used. On the other hand, the response of the various lymphocyte preparations from transplanted patients appeared less inhibited by steroids than the corresponding preparations from normal subjects. The addition to the culture of the adherent cell product interleukin 1 was effective in removing to a similar extent the steroid inhibitory effect on lymphocytes from normal and transplanted subjects. Thus, the lesser inhibitory effect of glucocorticoids on transplanted patient lymphocytes could be explained by the higher percentages of monocytes present in all peripheral blood mononuclear cell preparations. These results suggest that during immunosuppressive therapy with glucocorticoids and azathioprine the residual circulating lymphocytes have a responsiveness to in vitro dexamethasone suppression similar to that of normal peripheral blood lymphocytes.     

Reactivity of ovine lymphocytes to phytohaemagglutinin and pokeweed mitogen during pregnancy and in the immediate post-parturient period.

Lymphocytes from sheep in late pregnancy and at parturition showed markedly impaired proliferative responses to phytohaemagglutinin (PHA) in vitro when cultures were supplemented with foetal bovine serum (FBS), as compared to the responses of lymphocytes from non-pregnant sheep, sheep at 40 days of gestation and sheep at 80 days of gestation. Similar responses to PHA were observed when the medium was supplemented with autologous plasma (AP), although the responses were of a lower order. In both cases elevated responses to PHA were apparent at 10 days post-parturition. The response with FBS was more marked than with AP. Progressive reduction of lymphocyte responses to pokeweed mitogen (PWM) in the presence of FBS and AP were less obvious, although it was still apparent that responses to PWM were depressed at 120 days of gestation and at parturition, when compared with lymphocyte responses during early pregnancy (at 40 days and 80 days of gestation). The difference was much more apparent with AP than with FBS and responses during early pregnancy were markedly higher than those with FBS. An increase in lymphocyte responsiveness to PWM 10 days post-parturition was evident whether FBS or AP was incorporated in the cultures. The response with FBS was again more marked than with AP.     

Immune depression in trypanosome-infected mice. I. Depressed T lymphocyte responses.

Using a wide range of experimental conditions, several kinds of T lymphocyte responses in spleen cell populations from trypanosome-infected mice were studied. Lymphocyte stimulation after culture with the mitogen concanavalin A or with histoincompatible cells differing at H-2 or minor lymphocyte-stimulating loci was reduced or abolished in spleen cells from infected mice when compared with responses of spleen cells from uninfected controls. In addition, cytotoxic lymphocytes were not generated in mixed lymphocyte cultures which contained spleen cells from infected animals. Allogeneic skin grafting experiments performed with normal and infected mice showed that a decreased T lymphocyte response also occurs in vivo. The depressed immune responses were not simply due to low numbers of T lymphocytes in spleens of infected animals, but reflected a generalized immune depression which was not antigen-specific.     

Inhibition of anti-CD3 and interleukin-2 stimulated T lymphocyte proliferation by peptidomimetic opioid compound

In continuation to our earlier studies with peptidomimetic opioid compounds, we have further investigated immunosuppressive properties of one of our peptidomimetic compound (Tyr-NH-CH2-CH2-O-Phe-NH2) using peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Peptidomimetic compound was evaluated for its effect on anti-CD3 and recombinant human interleukin-2 (rhIL-2) stimulated lymphocyte proliferation in vitro and lipopolysaccharide (LPS) induced activation of mitogen activated protein kinase (MAPK, pp42/44) in mouse macrophage cells (RAW 264.7). Our results show the immunosuppressive potential of synthetic peptidomimetic compound. This compound significantly inhibited anti-CD3 and rhIL-2 stimulated lymphocyte proliferation in vitro. However, this peptidomimetic compound did not show any effect on LPS induced MAPK activation. These observations suggest that above peptidomimetic compound has potential to inhibit immune responses mediated by lymphocytes.     

Marijuana and immunity: tetrahydrocannabinol mediated inhibition of lymphocyte blastogenesis

The effects of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-OH delta-9-tetrahydrocannabinol (11-OH THC) on mitogen driven lymphocyte blastogenic transformation (LBT) were studied. THC inhibited LBT responses to the T lymphocyte mitogens phytohemagglutinin and concanavalin A but not the B lymphocyte stimulant pokeweed mitogen. The metabolite 11-OH THC caused a comparable, but less significant, inhibition of LBT responses to the T cell mitogens. These inhibitions were dependent upon the drug doses and the time of incubation with the lymphocytes. There was no significant inhibitory activity of THC to the LBT when it was added 24 or 48 h after mitogen. In addition, exposure of lymphocytes to THC for 3 h, followed by removal of the drug prior to addition of mitogen had no effect on the cells' ability to respond to the mitogen. Thus, there appears to be a specified temporal period during which exposure of lymphocytes to THC results in an inhibition of blastogenesis. Interleukin 2 (30 units/ml) could not preclude the THC induced inhibition of lymphocyte blastogenesis. We conclude that THC and 11-OH THC inhibit T lymphocyte blastogenesis. However, unlike the THC mediated inhibition of natural killer cell activity (as shown previously), the process is not responsive to the cytokine interleukin 2.     

The immunosuppressive potency of various steroids on peripheral blood lymphocytes, T cells, NK and K cells

The immunosuppressive effect of natural and synthetic steroids was tested in vitro on phytohemagglutinin (PHA) stimulated T lymphocytes and peripheral blood lymphocytes (PBL), as well as on NK and K cell activity. Three groups of steroids, significantly different in their immunosuppressive activity, were identified. Fluorohydrocortisone, and methylprednisolone were highly potent in suppressing PHA stimulation of T lymphocytes and PBL. Hydrocortisone was of intermediate potency, whereas cortisone, dihydrocortisol, and tetrahydrocortisol were of low potency. T lymphocytes were more sensitive to the suppressive effect of fluorohydrocortisone and methylprednisolone than were PBL cultures. NK and K cell activity was suppressed only by the high potency synthetic steroids and even then the suppression of K cell activity was not significant except at high in vitro steroid concentrations. The present findings support the conception that different lymphocyte subpopulations have different susceptibility to the effect of highly potent steroids. Thus, lymphocyte heterogeneity must be taken into account when the immunosuppressive potencies of different glucocorticoids are studied. Furthermore, the findings in different lymphocyte populations ranked the relative in vitro immunosuppressive potency of glucocorticoids different from the relative anti-inflammatory potencies reported in the literature.     

Enhancement of lymphocyte blastogenic and delayed hypersensitivity skin responses by indomethacin.

Incubation of Mycobacterium bovis-sensitized bovine peripheral blood lymphocytes with concanavalin A or M. bovis purified protein derivative and indomethacin caused a consistent, statistically significant increase in [3H] thymidine uptake as compared to cultures without indomethacin. The kinetics of the response showed that indomethacin must be added to lymphocyte cultures within hours after mitogen or antigen addition for enhancement of [3H]thymidine uptake to occur. Lymphocyte blastogenic responses to purified protein derivative were enhanced in both normal and M. bovis-sensitized lymphocyte cultures. However, enhancement of sensitized lymphocyte responses was significantly (P less than 0.01) greater than that in normal animals. Additionally, indomethacin treatment of M. bovis-sensitized guinea pigs singnificantly augmented delayed skin reactions to tuberculin. Delayed hypersensitive skin reactions were only enhanced when indomethacin was administered simultaneously with tuberculin.