Effects of interleukin-1 alpha and cyclosporin A in vivo and in vitro on bone and lymphoid tissues in mice

The purpose of this study was to investigate the effects of interleukin-1 alpha (IL-1 alpha) infusion and the ability of cyclosporin A (CYA) to alter IL-1 alpha-induced effects on bone in vivo and in vitro and lymphoid organs in vivo. Mice were administered: IL-1 alpha (2, 4, or 6 days), CYA (6 days), or IL-1 alpha and CYA (6 days). Hypercalcemia was induced in mice treated with IL-1 alpha compared to controls and CYA treated mice, and decreased urinary calcium excretion was present in IL-1 alpha and CYA groups. Osteoclastic bone resorption was increased with a resultant loss of total bone area and bone formation (as measured by mineral apposition rate) was decreased in mice infused with IL-1 alpha. Although CYA-treatment increased bone formation as compared to IL-1 alpha-treatment; CYA in combination with IL-1 alpha did not alter the reduction in mineral apposition rate caused by IL-1 alpha, IL-1 alpha also stimulated bone resorption in vitro which was significantly inhibited by cyclosporin A. IL-1 alpha-induced splenic granulopoiesis, peripheral blood neutrophilia, thymic atrophy, and lymphoid hyperplasia in lymph nodes. CYA-treatment resulted histologically in a severe depletion of lymphocytes in the thymus, a moderate depletion of lymphocytes in lymph nodes but no difference in the histology of the spleen compared to controls. In summary, interleukin-1 alpha was effective in stimulating hypercalcemia and bone resorption both in vivo and in vitro but cyclosporin A was effective in inhibiting IL-1 alpha-mediated bone resorption only in vitro. IL-1 alpha also had marked effects on spleen, thymus, and circulating blood cells; however, most parameters were not affected by the concurrent administration of cyclosporin A.     

In vitro and in vivo induction of tumor necrosis factor alpha by Borrelia burgdorferi.

Tumor necrosis factor alpha (TNF-alpha) is an immunoregulatory cytokine with many biological activities including the mediation of inflammation. We examined sera and synovial fluids from patients seropositive for infection with Borrelia burgdorferi using a radioimmunoassay specific for TNF-alpha. Significant elevation of TNF-alpha was found in the sera and synovial fluids of patients examined, while controls showed no elevation. Sera of mice infected with B. burgdorferi contained elevated levels of TNF-alpha which varied during the course of a 24-day infection. To determine whether B. burgdorferi is capable of inducing TNF-alpha production, spirochetes were added to adherent human peripheral blood mononuclear cells or mouse peritoneal exudate cells and 24 h later supernatants were assayed. TNF-alpha induction occurred in a dose-dependent manner. The maximum stimulation occurred when a ratio of 1 to 10 spirochetes per mononuclear cell was used. At optimal concentrations, induction was not diminished by inactivation of spirochetes or pretreatment with polymyxin B. These results suggest that an increase in TNF-alpha production may occur as a result of infection with B. burgdorferi.     

The interleukin-2 receptor in human monocytes and macrophages: regulation of expression and release of the alpha and beta chains (p55 and p75).

Human monocytes constitutively express the intermediate-affinity interleukin-2 receptor (IL2R) p75, while freshly isolated monocytes lack the low-affinity IL2R p55 (Tac antigen, CD25). Lipopolysaccharide (LPS) upregulates expression of p75 and effectively induces surface expression of CD25 on human monocytes within 18 h, as detected by two-colour FACS analysis on 59.5 +/- 7.6% of cells. IL2-binding studies using biotinylated IL2 reveal the presence of a functional high-affinity receptor on LPS-activated monocytes. Soluble CD25 (sCD25) is not released into supernatants of elutriation-purified monocytes cultured for 24 h with 100 ng/ml LPS. When these monocytes are cultured for up to 7 days in the presence of serum to induce differentiation into macrophages, increasing amounts of sCD25 can be measured in the 24-h supernatants induced with LPS (173 +/- 86 U/ml at day 7), whereas the percentage of CD25+ cells (14.8 +/- 14.1% at day 7) is significantly lower than in monocytes. Thus, the cell surface expression and release of CD25 is differentially regulated in activated monocytes and macrophages.     

Expression of interleukin-2R alpha and interleukin-2R beta in Hodgkin's disease.

Hodgkin and Reed-Sternberg cells, the putative malignant cells of Hodgkin's disease (HD), carry regularly the CD25 antigen that forms one chain of the interleukin-2 (IL-2) receptor (IL-2R alpha). To analyze the putative role of IL-2R expression in Hodgkin's disease, we have investigated the expression of both IL-2R alpha and IL-2R beta chains in HD-derived cell lines and in primary specimens from patients with HD. Expression of IL-2R alpha and IL-2R beta was detected in all HD-derived cell lines. In addition, soluble IL-2R alpha molecules were demonstrated in the supernatants of three of these cultured cell lines. In primary tissues, IL-2R alpha and IL-2R beta were seen in some but not all cases. Staining was detected in Hodgkin and Reed-Sternberg and in lymphoid cells. There was a remarkable difference in the pattern of expression, in that IL-2R alpha- but not IL-2R beta-positive cells from HD patients were clustered in frozen sections. We conclude from these data that IL-2R expression might be involved in the biology of HD.     

Anti-CD3 antibody-induced expression of both p55 and p75 chains of the high affinity interleukin-2 receptor on human T lymphocytes is inhibited by cyclosporin A.

The inhibitory effect of cyclosporin (CsA) was investigated on human lymphocytes stimulated by anti-T-cell antibodies (anti-CD3 and -CD2) or mitogenic lectins. Whereas inhibition of cell proliferation (50%) occurred at 10 ng/ml CsA after cell activation via CD3 or CD2, higher CsA concentrations (300 ng/ml) were necessary to inhibit lectin-mediated cell activation (PHA, Con A). Exogenous recombinant interleukin-2 (rIL-2) partially reversed the inhibitory effect on antibody-stimulated cells only; however, at higher CsA concentrations (300 ng/ml) proliferation was again inhibited. Thus, CsA affected IL-2R expression and/or function at higher concentrations (300 ng/ml). CsA had no effect on receptor function as measured on IL-2-dependent cell growth of CTLL cells or preactivated lymphocytes. However, CsA inhibited both high and low affinity receptor expression as shown by [125I]IL-2 equilibrium binding studies on anti-CD3-stimulated cells. Cross-linking studies revealed that both p55 (TAC) and p75 chains of the IL-2R were not induced at low CsA concentrations (10 ng/ml). However, addition of rIL-2 reversed CsA inhibition of IL-2R expression. It is concluded that CsA, at least in anti-CD3-stimulated cells, inhibits IL-2R expression and cell proliferation with similar potency. Exogenous rIL-2 reverses CsA inhibition of IL-2R expression. This might be due to binding of rIL-2 to receptors which escape CsA inhibition, thereby up-regulating receptor expression which is drug resistant.     

Preferential association of alpha and beta chains of the T cell antigen receptor.

The cytotoxic T lymphocyte (CTL) clone B6.2.16 expresses a V beta 8.2/J beta 2.3/C beta 2-encoded T cell receptor (TcR) beta chain and an alpha chain that is encoded by a novel V alpha gene segment, J alpha 27 and C alpha. While expression of V alpha B6.2.16 and J alpha 27 is not detectable in lymph node cells of normal C57BL/6 mice, expression of these gene segments was readily seen in transgenic mice expressing the rearranged beta chain gene of the B6.2.16 T cell clone. This finding indicates that only a limited number of alpha chains can associate with the B6.2.16 beta chain and strongly suggests that the size of the TcR repertoire of mature T cells is not only limited by TcR ligand-mediated thymic selection but also by restrictions in alpha-beta combinatorial chain association.     

In vivo and in vitro action of chloroquine on surface markers of human peripheral lymphocytes.

Chloroquine was administered orally to twenty normal individuals and the effect of the drug on surface markers of peripheral bloof lymphocytes was studied. The total number of circulating lymphocytes and leucocytes in the blood did not change significantly after chloroquine administration. However, there was a significant fall in the percentage and number of lymphocytes with erythrocyte (E) and C'3 markers and an increase in cells lacking both these markers. In vitro experiments were carried out to study the mechanism of action of the drug on the expression of the lymphocyte receptors. Lymphocytes treated with chloroquine in vitro failed to show any change in their capacity to bind erythrocytes or erythrocytes coated with Ab and complement. The sera from chloroquine-treated individuals failed to show any factor inhibiting E and EAC rosette formation. The studies indicate that chloroquine may not act directly on the lymphocyte surface markers and cause inhibition of their expression but that it may act in some indirect way affecting one or more of the many factors involved in the normal expression of the markers.     

Inhibition by cyclosporin A of rodent malaria in vivo and human malaria in vitro.

The development and course of normally lethal parasitemias in mice inoculated intraperitoneally with erythrocytic stages of Plasmodium yoelii or Plasmodium berghei were markedly affected by treatment with the antilymphoid drug cyclosporin A (CS-A). When the first of four daily subcutaneous 25-mg/kg doses of CS-A was given at the time of parasite inoculation, patent infections failed to develop. If begun up to 5 days earlier, this same treatment regimen prolonged the prepatent period, attenuated parasitemia, and reduced mortality. In mice with patient infections, two consecutive daily 25-mg/kg doses of CS-A were sufficient to terminate parasitemias which, after several days, reappeared but were self-limiting. This pattern of apparent cure followed by transient recrudescence remained unaltered even when daily treatment with the same drug dose was continued for 3 weeks. Recrudescence was associated with the emergence of parasite populations that were relatively resistant to CS-A and, in the case of P. yoelii, of reduced virulence. In more limited experiments, CS-A was found to be active in vitro against erythrocytic stages of the human malarial parasite palsmodium falciparum. Depending on the concentration of drug in the culture medium, parasite growth was either prevented or inhibited.     

Effect of swainsonine on interleukin-2 alpha chain receptor expression and proliferation of human lymphocytes.

Swainsonine, an inhibitor of mannosidase II, involved in N-linked glycoprotein processing, modifies expression of cell surface receptors. This alkaloid has strong anti-metastatic and immunomodulatory activity; it enhances stimulation of lymphocytes triggered by concanavalin A (ConA) but suppresses stimulatory effects of phytohaemagglutinin (PHA). We presently observe that swainsonine decreases expression of the interleukin-2 (IL-2) receptor (IL-2R) on PHA-stimulated human peripheral blood lymphocytes, measured by binding of a monoclonal antibody that recognizes the 55-kD glycoprotein subunit (alpha) of this receptor. Proliferation of the PHA-stimulated lymphocytes is suppressed by swainsonine, which manifests in the decreased proportion of both cells entering G1 (from G0) and those progressing through S. G2 and M. This suppression can be overcome by addition of IL-2 into cultures. In contrast, swainsonine has no effect on IL-2R expression and stimulation (cell cycle progression) of lymphocytes triggered by the monoclonal antibody OKT3. The data suggest a possibility that the observed swainsonine effects on lymphocytes stimulated by PHA are mediated via surface receptors other than IL-2R. These receptors may appear prior to IL-2R and be also involved in cell stimulation by PHA but not by other mitogens.     

T cell subset-specific expression of antigen receptor beta chains in alpha chain-transgenic mice.

A large number of V beta 8 gene-encoded cDNA were analyzed from peripheral CD4+ and CD8+ T cell subsets of T cell receptor (TcR) alpha chain-transgenic mice. This analysis demonstrates that a limited repertoire of TcR beta chains are co-expressed with the transgenic alpha chain. Most importantly, certain V beta 8-J beta combinations were found exclusively in one of the subsets and, in some cases, subset-specific differences were localized to the VDJ junctional region of the beta chain genes. In contrast, CD4-CD8- transgenic T cells, as well as CD4+ and CD8+ T cells from normal littermate controls, were found to express diverse beta chain repertoires. The present study suggests that beta chains with distinct structural characteristics are expressed in the CD4+ and CD8+ subsets, respectively. Moreover, the data suggest that the same structural constraints do not apply to the population of CD4-CD8- transgenic T cells.     

Requirements for the induction of the interleukin-2 receptor complex in a human pre-T-cell line.

Cell line PER-117 is a T-cell receptor negative human T-cell line that can be induced to express a functional interleukin-2 receptor (IL-2R). Recombinant interleukin-1 (IL-1) as well as certain combinations of inducer substances could be shown to stimulate the expression of the p55 (alpha)-chain of the IL-2R in PER-117 cells. The synergistic increases in IL-2R alpha expression were demonstrated at the cell surface as well as at the mRNA level. The results suggested that in PER-117 cells IL-1 appears to induce expression of the alpha-chain by pathways that are different to activation via protein kinase C (PKC), and that drug-induced cyclic AMP (cAMP) activation did not substitute for IL-1. We found that the regulation of mRNA for IL-2R beta (p75) differed significantly from that seen for IL-2R alpha. Moreover, the requirements for IL-2R alpha induction determined for this cell line differ from other human cell lines, which may reflect that there are distinct requirements for activation depending on the stage of differentiation and/or lineage of the cells. The PER-117 cell line provides a unique model to examine further the mechanism leading to induction of a functional IL-2R at an early stage of human T-cell differentiation.     

In vitro and in vivo bioactivity of single-chain interleukin-12

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.     

Effects of experimental conditions on the production of interleukin-1 alpha and -1 beta by human endothelial cells cultured in vitro.

We have characterized the production of IL-1 alpha and -beta in primary and passaged cultures of quiescent human umbilical vein endothelial cells (HUVECs) using highly specific and sensitive solid-phase enzyme immunoassays. Primary cultures produced both immunoreactive IL-1 alpha and IL-1 beta following stimulation with lipopolysaccharide with the alpha form predominating over the beta. Most of the IL-1 produced remained cell-associated. Primary, but not passaged, cultures were significantly contaminated by macrophage-like cells, possibly accounting for higher production of IL-1, especially IL-1 beta. Gel filtration of secreted proteins derived from cultured HUVECs showed that the immunoreactive IL-1 alpha exhibited the expected molecular weight (17 kDa), but cell-associated IL-1s appeared to be a mixture of the 17 kDa protein and of higher molecular weight precursors. Mitogens in the culture medium (serum and endothelial cell growth supplement) were powerful stimuli of endothelial IL-1 production and accounted for the relatively high basal IL-1 levels observed in the cultured endothelial cells. The proliferative phenotype of the endothelium is possibly linked to the expression of high level of IL-1, which until now was thought to be an autocrine inhibitor of endothelial cell mitosis.     

Interleukin-1 alpha and interleukin-1 beta in preterm and term human parturition.

Interleukin-1 (IL-1) has been implicated in the mechanism of human parturition in the setting of infection. The purpose of this study was to determine the effect of labor (term and preterm) and microbial invasion of the amniotic cavity on amniotic fluid (AF) concentrations IL-1 alpha and IL-1 beta. AF was retrieved by transabdominal amniocentesis from the following groups of women: midtrimester genetic amniocentesis (16 to 18 wk) (N = 15), preterm labor with intact membranes (21 to 36 wk) with or without infection (N = 72), preterm premature rupture of membranes (PROM) (N = 88), and term not in labor or in active labor with or without infection (N = 58). AF was cultured for aerobic and anaerobic bacteria as well as Mycoplasmas. IL-1 was measured with a commercially available immunoassay validated for AF (sensitivity: IL-1 alpha, 157 pg/ml; IL-1 beta, 50 pg/ml). All women at midtrimester had undetectable AF IL-1 alpha and IL-1 beta. Among women in preterm labor with positive AF cultures, IL-1 alpha and IL-1 beta were detectable in the AF in 86.6% (13/15) and 100% (15/15), respectively. In contrast, all women with negative AF cultures without labor (N = 36) had undetectable AF IL-1 alpha concentrations and 52.7% (19/36) had undetectable AF IL-1 beta concentrations. Histopathological chorioamnionitis was present in 92.8% (13/14) of patients who had positive AF cultures and detectable IL-1 in the AF. IL-1 was significantly higher in patients with preterm PROM, labor, and positive AF cultures than in the other subgroups of patients with preterm PROM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes

Thymosin alpha 1 (T alpha 1) and thymosin fraction 5 (TF5) have been shown to induce lymphocyte maturation and differentiation as well as to modulate mature immune responses to antigens and mitogens. The present study focused on the characterization of the mechanisms involved in T alpha 1 and TF5 enhancement of phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) secretion and interleukin-2 receptor (IL-2R) expression in human mononuclear cells. We provide evidence that TF5 and T alpha 1 modulate an early event(s) during lymphocyte activation by mitogens. A short preincubation period (30 min) of non-adherent cells with thymosins, followed by extensive washing and subsequent exposure to PHA, was sufficient to enhance the production of IL-2 and the expression of IL-2R induced by the mitogen. Furthermore, the concomitant addition of PHA and thymosin during the preincubation period is not necessary for the enhancing effects to occur. We have also studied the role of macrophages on thymosin modulation of these responses. Results presented here indicate that macrophages are not essential for the interaction of thymosins with T-cells. However, macrophages are an absolute requirement during the exposure to the mitogen after preincubation with thymosins for the manifestation of TF5- and T alpha 1-mediated enhancing effects on IL-2 production and IL-2R expression. Human recombinant interleukin-1 beta (rIL-1 beta) was able to replace this macrophage requirement, indicating that production of IL-1 by these cells is a critical event in thymosin modulation of the IL-2 system. Two-color flow cytometric analysis and experiments involving the use of highly purified helper/inducer (Th, CD4+) and cytotoxic/suppressor (Tc, CD8+) T-cell populations indicated that both, Th and Tc cell populations are targets of thymosin activity. These studies provide additional evidence that thymosins play an important role in the modulation of the normal immune response and begin to define the mechanisms underlying T alpha 1 immunoregulatory properties.     

In vivo elimination of T cells expressing specific T-cell receptor V beta chains in mice susceptible to collagen-induced arthritis.

Type II collagen-induced arthritis (CIA) is believed to be dependent on T cells expressing a limited number of V beta chains. Two different methods were used to selectively eliminate T cells expressing a certain T-cell receptor (TcR) V beta chain in mouse strains susceptible to CIA. In vivo treatment with monoclonal anti-V beta 6 or anti-V beta 8.1,2 antibodies did not alter CIA, despite a reduction of the major part of the V beta 6+ or V beta 8.1,2+ lymph node cells (LNC), as measured by flow cytometric (FACS) analyses. The reduction was not due to complete elimination of V beta 6+ or V beta 8.1,2+ cells, since part of the V beta 6 and V beta 8.1,2 expressing cells returned later, even in mice that had been thymectomized first to prevent maturation of new T cells. In contrast, treatment with antibodies against CD4 efficiently abrogated development of CIA. In the (CBA x DBA/1J)F1 and the (BALB/c x DBA/1J)F1 mice, where M1s1a was combined with expression of I-E, the V beta 6+ LNC were deleted. In spite of the deletion, both F1 strains were highly susceptible to CIA.     

In vivo beneficial effects of cyclosporin A and 1,25-dihydroxyvitamin D3 on the induction of experimental autoimmune thyroiditis

In a recent work, we provided evidence that the in vitro inhibitory effect of cyclosporin A (CsA) was potentiated by the addition of another immunosuppressive molecule, the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In the present study, we investigated the in vivo influence of the association of both drugs administered at infratherapeutic doses, using an experimental model of autoimmune thyroiditis in CBA mice. Treatment regimen of the animals was initiated at priming with thyroglobulin (Tg) and consisted of daily administration of CsA (10 and 20 mg/kg/day, intragastrically) and/or 1,25(OH)2D3 (0.1 and 0.2 microgram/kg/day, ip) for 21 days. Control mice that were given a placebo preparation orally and the vehicle of vitamin D3 metabolite ip developed a severe disease as assessed by histological examination on Day 28 postimmunization and detection of circulating anti-Tg antibodies. Treatment with either drug administered alone at the doses mentioned above did not affect the incidence of thyroiditis and only reduced by up to 26% the severity of histological lesions. In contrast, the mice treated simultaneously with both drugs exhibited a lower incidence of thyroid pathology and developed a significantly milder disease (P less than 0.001) as compared to controls. However, there was no alteration in the levels of anti-Tg antibodies. This in vivo beneficial effect of low doses of CsA and 1,25(OH)2D3 was not due to an accumulation of CsA in the blood of treated mice since the levels of CsA were similar, regardless of the administration of 1,25(OH)2D3. Our data suggest that these two immunomodulatory agents used together at low doses may be an effective therapy of autoimmune disorders with fewer side effects.