Jana Raskova

IMMUNE DEFICIENCY IN UREMIA : INTERLEUKIN-2 PRODUCTION AND RESPONSIVENESS AND INTERLEUKIN-2 RECEPTOR EXPRESSION AND RELEASE

We have studied the role of interleukin-2 (IL-2) and its receptors in the impaired in vitro lymphocyte response characteristic of hemodialysis patients treated by means of cuprophane membranes. The proliferative response of T lymphocytes as well as T-cell-dependent B cell proliferation after stimulation with mitogens was significantly reduced in hemodialysis patients. The in vitro production of IL-2 after such stimulation in parallel cultures was found to be similar in patients and in controls. The expression of IL-2 receptor on the lymphocyte cellular membrane in the hemodialysis group was also similar to controls. The in vitro proliferative response of uremic lymphocytes to exogenous IL-2, however, was significantly depressed suggesting a reduced availability of biologically active IL-2 receptor. The release of soluble IL-2 receptor by lectin-stimulated lymphocytes in culture was also significantly lower in the patient group; yet, hemodialysis patients has a strikingly elevated level of plasma soluble IL-2 receptor, and similar high levels were also found in three other groups of end-stage renal disease patients dialyzed by means of cellulose acetate, polysulfone and polyacrylonitrile membranes, as well as in a group of uremic patients on conservative treatment. In the hemodialysis patient group a significant positive correlation between levels of soluble IL-2 receptor and the duration of hemodialysis was found. Since soluble IL-2 receptor has been reported to down-regulate lymphocyte responses, the elevation in plasma levels of soluble IL-2 receptor in hemodialysis patients may be a pathogenetic factor in the progressive development of impaired immunity associated with end-stage renal disease.

Soluble and cellular markers of immune activation in patients with systemic sclerosis

The peripheral blood lymphocyte pattern, the lymphocyte responses in vitro, as well as the soluble markers of immune activation were studied in 24 patients with systemic sclerosis (SSc patients). The proportions of total T cells (CD3), their CD4 subset, as well as B lymphocytes were within the normal range. The relative proportion of CD8 lymphocytes, however, was significantly reduced. Patients with SSc had a slightly lower percentage of CD4/4B4+ cells, whereas their proportion of CD4/2H4+ cells was elevated as compared to healthy controls. The proportion of lymphocytes expressing the interleukin-2 receptor (IL-2R) was significantly higher in SSc patients. The proliferative responses of peripheral blood mononuclear cells to PHA stimulation were reduced in the patient group, while expression of IL-2R on lymphocytes after such in vitro stimulation was comparable to that of controls. Expression of IL-2R on patient but not control lymphocytes was increased after in vitro exposure to laminin. Such exposure failed to induce IL-2 production or cell proliferative responses. Soluble plasma IL-2R level (sIL-2R) and soluble CD8 (sCD8) molecule levels in SSc patients were significantly elevated. These results indicate the presence of an ongoing lymphocyte activation in this disease process.

Effects of cyclosporine and rapamycin on immunoglobulin production by preactivated human B cells

In order to assess the direct effects of cyclosporine A (CsA) and rapamycin on B cells, we utilized a two‐segment culture system of highly purified B lymphocytes consisting of induction (activation) in the presence of the formalinized Staphylococcus aureus bacteria and IL‐2. and differentiation, respectively, in the presence of various combinations of cytokines. Results show that rapamycin strongly inhibited production of both IgM and IgG measured at the end of the secondary culture supported by IL‐2/IL‐6, whereas CsA up‐regulated the immunoglobulin production. The stimulatory effect of CsA was also observed when preactivated B cells were recultured in absence of any cytokines. These results show that rapamycin and CsA have dearly distinct effects on human B lymphocyte responses in vitro. Rapamycin is a more potent in vitro immunosuppressant of B lymphocytes than CsA. It is effective at significantly lower concentrations, and it does not stimulate either the proliferation or antibody production by preactivated B cells.

IL-2 responsiveness of lectin-induced lymphoblasts: soluble IL-2 receptor release and differential in vitro effects of immunosuppressants.

We examined the mode of action of different immunosuppressants on the responsiveness of phytohemagglutinin (PHA)-induced lymphoblasts further stimulated by recombinant interleukin-2 (rIL-2). The stimulation of PHA blasts with rIL-2 resulted in an enhancement of tritiated thymidine ([3H]TdR) incorporation and of soluble interleukin-2 receptor (sIL-2R) release. Cyclosporin A (CsA) and prednisolone inhibited in different ways the responsiveness of PHA pre-stimulated blood mononuclear cells (PBMC) to rIL-2, as measured by [3H]TdR incorporation. The addition of CsA resulted in considerable enhancement of the release of sIL-2R, whereas the addition of prednisolone was associated with a similar enhancement only when the higher concentrations of rIL-2 were employed. EGTA, a calcium (Ca2+) chelator, and verapamil, a Ca2+ channel blocker, inhibited [3H]TdR incorporation in a concentration-dependent manner. EGTA inhibited sIL-2R release in the same manner when used alone, and reversed the CsA- and prednisolone-induced enhancement of sIL-2R release by rIL-2 induced lymphoblasts, when used in combination with CsA or prednisolone. Verapamil had a similar but less striking effect. The effects of CsA and prednisolone were also studied in PHA-induced blasts originating from purified CD4+ or CD8+ lymphocytes. Stimulation of these blasts with rIL-2 resulted in higher [3H]TdR incorporation by CD8+ blasts than by CD4+ blasts: however, no sIL-2R release was detected in supernatants of either CD4+ or of CD8+ blasts. Both CsA and prednisolone inhibited the rIL-2-induced enhancement of [3H]TdR incorporation by both T-cell subsets.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and Kinetics of Interleukin-1 in Hemodialysis

Interleukin-1-beta (IL-1-beta) was measured in the plasma and peripheral blood mononuclear cell lysates of uremic patients undergoing maintenance hemodialysis by means of either cuprophane or polysulfone membranes. Basal plasma levels of IL-1-beta in hemodialyzed patients were strikingly higher than those of uremic patients on conservative treatment or of healthy subjects. Plasma levels of IL-1-beta in uremic patients increased significantly after 3 and 6 months of hemodialysis. The study of the kinetics of IL-1-beta concentration during a single hemodialysis session revealed that the concentration of IL-1-beta fell to 21 and 22% of the predialysis level with cuprophane and polysulfone, respectively. Hemodialysis patients also had a significantly higher intracellular IL-1-beta level than normal controls. During the hemodialysis session, an increase in cell-associated IL-1-beta was seen regardless of the membrane employed. In a parallel study, normal mononuclear cells were subjected to closed-loop in vitro dialysis with either cuprophane or polysulfone membranes, with or without acetate buffer. After 120 min of recirculation, an increase in cell-associated IL-1-beta was detected, but no changes were seen in the circulating medium. IL-1-beta production was not significantly influenced by either membrane or the dialysate composition. Hemodialysis has been associated with high plasma- and cell-associated IL-1 levels. The kinetics of intradialytic changes of IL-1-beta levels make IL-1 an unlikely cause of acute complications in hemodialysis. On the other hand, a chronic elevation of IL-1 in plasma of patients on maintenance hemodialysis may contribute to the development of some of the long-term complications of this treatment.

Cyclosporine A and prednisolone inhibit lectin- and alloantigen-induced release of sCD8: Correlation with proliferative responses

It has been shown previously that there is a strong correlation between the in vitro release of soluble CD8 glycoprotein (sCD8) and CD8+ T lymphocyte activation. In the present study, the lectin stimulation of peripheral blood mononuclear cells (PBMC) induced a dose-dependent release of sCD8 which correlated with the magnitude of CD8 lymphocyte activation as measured by the expression of the interleukin 2 (IL-2) receptor and HLA-DR antigen and of the T cell proliferative responses. Both the proliferative responses and the release of sCD8 were inhibited by cyclosporine A (CyA) and prednisolone (PRED) in a concentration-dependent manner. When the immunosuppressants were present for only 60 min before the initiation of the cultures, an inhibitory effect was also seen, but this was maximal only when the agents were added at the initiation of the culture period; when the addition of CyA or PRED was delayed for either 24 or 48 hr after the initiation of the culture, the degree of inhibition of the proliferative response was greatly reduced. However, there was a significant inhibition of sCD8 release by CyA even when it was added 48 hr after the culture initiation. The addition of recombinant IL-2 did not affect the lectin-induced sCD8 release. The inhibition of the lectin-induced proliferative response and sCD8 release by PRED, but not that by CyA, was reversed by the recombinant IL-2. Alloantigen stimulation also induced sCD8 release and this release was inhibited both by CyA and by PRED. These data, together with the known effects of CyA on differentiation, clonal amplification, and activation of CD8 T lymphocytes, suggest that in vitro sCD8 release occurs during the early stages of activation of CD8+ cytotoxic T cells.

Adenovirus type 12 tumor antigen I. Separation from DNA polymerase α and immunoprecipitation of tumor-antigen polypeptides

Abstract Adenovirus type 12 (Ad12) tumor (T) antigen, extracted from Ad12-transformed hamster cells and partially purified (>500-fold) by chromatography on double-stranded DNA-cellulose, contains DNA polymerase α activity which is partially inhibited by antibodies reacting with T antigen. The DNA polymerase and T antigen can be completely separated by chromatography on single-stranded DNA-cellulose to which T antigen does not bind. Antibodies reacting with T antigen have no inhibitory effect on the isolated DNA polymerase. The fraction eluting in the void volume of a single-stranded DNA-cellulose column containing >1200-fold purified T antigen stimulates markedly the activity of DNA polymerase α with “native” DNA template; with the “activated” DNA template the stimulation is less prominent. This stimulation is not significantly affected by T antigen-reactive antibodies. An analogous fraction of control BHK21 cells has similar stimulatory effects on the isolated DNA polymerase α, indicating that the stimulatory activity is probably not attributable to T antigen. The T-antigen polypeptides were immunoprecipitated from >1200-fold-purified preparations by antibodies from tumor-bearing hamsters and were identified as a major peptide of molecular weight 50,000 and a minor species of molecular weight 11,000.

IL-4 receptor expression by SAC-activated B-lymphocytes: Its role in B-cell proliferation and the effect of cyclosporine (CsA), prednisolone and verapamil

Expression of the IL-4 receptor was studied in a highly purified population of human B-lymphocytes stimulated by Staphylococcus aureus, cowan I (SAC). Flow cytometric analysis showed that incubation with SAC in the absence of detectable levels of IL-2, IL-4 and IL-6 resulted in a striking increase in cellular binding of IL-4. The SAC-stimulated B-cells responded to exogenous IL-4 by DNA synthesis. This response was unaffected by CsA or prednisolone, but was inhibited by the Ca2+ channel blocker verapamil.

Effect of verapamil on the IL-2 binding to its active receptor and on the release of IL-2 receptor by activated PBMC

In the present study we have examined the effect of a Ca2+ channel blocker (verapamil) on the binding of IL-2 to its biologically active receptor (IL-2R), as well as on the release of its soluble form (sIL-2R) by peripheral blood mononuclear cells (PBMC) stimulated by a variety of stimuli. In the same culture systems, cyclosporine A (CsA) was also used as an additional dissecting tool. PHA and the Ca2+ ionophore A23187 enhanced both the percentage of PBMC binding phycoerythrin-conjugated IL-2 (PE-IL-2) and the mean fluorescence intensity of this binding. A phorbol-ester (PMA), on the other hand, enhanced only slightly the proportion of PE-IL-2 binding cells. The two stimulatory combinations (PHA/PMA and A23187/PMA) also up-regulated the proportion of PE-IL-2 binding cells and the fluorescence intensity; the PHA/PMA combination was the most potent of all stimuli used. These two stimulatory combinations, and PHA alone, were also associated with maximal in vitro release of sIL-2R. Verapamil significantly down-regulated PE-IL-2 binding in all culture systems and it convincingly inhibited the release of sIL-2R. Furthermore, this mode of action of verapamil was concentration-dependent. CsA, on the other hand, inhibited the binding of PE-IL-2 to all stimulant-activated PBMC and had only a slight inhibitory effect on the in vitro release of sIL-2R. Our results indicate that there is a correlation between the binding of IL-2 to biologically active receptors on the surface of stimulant-activated PBMC and the release of the soluble form of IL-2R by the same cells.(ABSTRACT TRUNCATED AT 250 WORDS)