Differential Inhibition by Cyclosporin A Reveals two Pathways for Activation of Lymphokine Synthesis in T Cells

Abstract

Two pathways for the activation of lymphokine synthesis in murine T cell clones and polyclonal T cell blast populations were identified. One was induced by ligands of the T cell receptor (TCR) and led to high production of GM-CSF, IFN-γ, and IL-3. The other was induced by IL-2 and led to production of lower levels of GM-CSF and IFN-γ with relatively little IL-3 synthesis. Cyclosporin A (CsA) markedly inhibited TCR-independent production of lymphokine mRNA and protein at concentrations where IL-2-dependent stimulation of lymphokine production and proliferation was unaffected. Stimulation of lymphokine synthesis by phorbol myristate acetate (PMA) and the Ca²⁺ ionophore ionomycin, or by ionomycin alone, mimicked the TCR-dependent response. PMA on its own was a preferential stimulus for GM-CSF production, but, whereas CsA did not inhibit PMA stimulation of polyclonal T cell blasts, T cell clones displayed a biphasic response in which CsA only inhibited stimulation by high PMA concentrations. The data suggest that Ca²⁺-independent (CsA-resistant) T cell activation induces synthesis of GM-CSF and IFN-γ but is a poor stimulus for IL-3 production. On the other hand, when Ca²⁺-dependent (CsA-sensitive) pathways are activated by TCR binding or by a Ca²⁺ ionophore, production of high levels of all three lymphokines can be induced.     

Competition of Chemically Related Antigens for Presentation by Accessory Cells to T Cells Requires Expenditure of Metabolic Energy by the Accessory Cells

The immune responsiveness of guinea pigs both to dinitrophenyl-poly-L-lysine (DNP-PLL) and to the lysine-rich random copolymer of L-glutamic acid and L-lysine (GL) is controlled by the 'poly-L-lysine gene'. We have previously demonstrated that accessory cells of responder strains can be made incapable of presenting DNP-PLL to responsive T cells in assays for proliferation, by in vitro exposure of the cells to GL before and during their exposure to DNP-PLL. We demonstrate here that the presence of anti-Ia antibody in the cultures does not interfere with the apparent competition of the two antigens for presentation by accessory cells. Furthermore, the two antigens do not compete for presentation when the accessory cells are exposed to them at 1 degree C, suggesting that endocytosis and/or other energy-requiring cellular events are necessary for the competition.     

Lymphokine production induced by streptococcal pyrogenic exotoxin-A is selectively down-regulated by pooled human IgG

The influence of pooled human IgG preparations for intravenous use (IVIg) on cytokine production induced by streptococcal pyrogenic exotoxin-A (SPE-A) was studied at the single-cell level using cytokine-specific monoclonal antibodies and indirect immunofluorescence or immunohistochemical staining. Mononuclear cells from healthy adult blood donors were stimulated with SPE-A alone or in the presence of IVIg. IVIg was added either prior to stimulation or 24 h after initiation of cultures, in an attempt to evaluate whether IVIg treatment could influence an already established systemic streptococcal disease. Cells were harvested after 48 or 72 h of culture and stained for the following cytokines: interleukin(IL)-1α, IL-1β, IL-1ra, IL-6, IL-8, IL-2, tumor necrosis factor interferon(IFN)-γ and TNF-α and TNF-β and granulocyte macrophage-colony-stimulating factor. Stimulation with SPE-A lead to extensive lymphokine and monokine production. With the addition of IVIg prior to stimulation there was a strong reduction of blast transformation and an almost complete inhibition of lymphokine production, in particular in the synthesis of IFN-γ and TNF-β while the synthesis of IL-1 and IL-8 was either unaffected or increased. Adding IVIg 24 h after SPE-A stimulation also resulted in reduced blast transformation and decreased synthesis of IFN-γ and TNF-β. These results indicate an immunomodulatory potential by IVIg on streptococcally induced T cell activation and lymphokine production.     

Abnormality of Spontaneous Lymphokine Synthesis by Lymphocytes of Patients with Connective Tissue Disorders

Forty-seven patients with various connective tissue disorders were studied to evaluate the spontaneous release of lymphocyte factors affecting the in vitro migration of guinea pig macrophages. In the assay used the lymphocytes from 8 patients produced an excessive amount of factors inhibiting macrophage migration while the lymphocytes from 12 patients produced an enhancement of migration. There were no differences in the delayed hypersensitivity skin test responses between the 2 groups of patients. The data are consistent with either an abnormality of suppressor lymphocyte function or an altered lymphocyte subpopulation relationship as a factor in this in vitro abnormality.     

Isolation of Substances Responsible for Lymphokine Activity from Sensitized Mouse Spleen Cells

Cultures of brucella-sensitized mouse spleen cells exposed to Brucella abortus antigens in vitro release macrophage migration inhibition factor (MIF) and macrophage spreading factor. Subjecting the supernatants from such cultures to preparative scale electrophoresis in acrylamide gel yields several fractions, one of which contains both MIF and macrophage spreading factor. This material has properties attributable to guinea pig MIF: it is nondialyzable, heat stable, nontoxic to macrophages from heterologous murine donors, and has a greater anodal electrophoretic mobility than guinea pig serum albumin. Another fraction from the gel column inhibits macrophage spreading; its electrophoretic mobility is similar to that of guinea pig gamma globulin. Neither brucella antigen nor skin reactive substances were detectable in any acrylamide gel column fraction when tested by the mouse footpad induration assay technique.     

Blastogenesis and Lymphokine Synthesis by T and B Lymphocytes from Patients with Periodontal Disease

Thymus-derived (T) and bone marrow-derived (B) lymphocytes were isolated from human peripheral blood and cultured with various mitogens and antigens. Purified protein derivative of tuberculin stimulated both purified T and B cells from patients with positive skin reactivity to purified protein derivative but did not stimulate nonimmune lymphocytes. Similarly, both T and B lymphocytes from patients with periodontal disease were stimulated to proliferate when incubated with dental plaque, whereas cells from normal individuals without gingivitis were unresponsive. In contrast, one component of plaque, bacterial endotoxins (lipopolysaccharide), minimally stimulated B lymphocytes from both normal or gingivitis patients. T lymphocytes from patients with periodontal disease were also stimulated by plaque antigen to produce chemotactic lymphokine activity (CTX) for human monocytes. B cells purified by the EAC rosetting method nonspecifically produced CTX without concomitant blastogenesis; however, after dissociation of adherent EAC these immune B cells did not spontaneously produce CTX. Lymphokine synthesis by B cells was not dependent on concomitant blastogenesis. Dissociated B cells from periodontitis patients also produced CTX activity after stimulation with dental plaque antigen. Therefore, both T and B lymphocytes, after stimulation with nonendotoxin antigenic components of plaque, proliferated and produced lymphokines, which are presumed to contribute to the pathogenesis of periodontal disease.     

Gamma-Interferon-Mediated Down-Regulation of Electrolyte Secretion by Intestinal Epithelial Cells: A Local Immune Mechanism?

Active chloride (Cl-) secretion by intestinal crypt enterocytes is the central pathophysiological disturbance in most cases of acute diarrhoea. We examined monolayers of the human intestinal cell line T84 mounted in Ussing chambers to see whether the T-cell lymphokine gamma interferon (IFN-gamma) might affect the Cl- secretory properties of these cells, which morphologically and functionally resemble native crypt enterocytes. Pretreatment of T84 cell layers with IFN-gamma for 24 h (but not for 3 h) markedly decreased the Cl- secretory response to vaso-active intestinal polypeptide (VIP) and to cholera toxin and carbachol without appreciably affecting the overall morphology, electrical resistance, or cyclic AMP response of the T84 cell monolayer. The IFN-gamma treatment, however, did induce subtle changes in the T84 cell membrane protein composition which might have affected ion channels regulating Cl- secretion. Our results may indicate a possible novel 'cell-mediated' immune mechanism through which activated gut T cells could modulate the extent of intestinal electrolyte and fluid secretion in, for example, enteric infections.     

E-Selectin: Sialyl Lewis, a Dependent Adhesion of Colon Cancer Cells, is Inhibited Differently by Antibodies Against E-Selectin Ligands

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe^x)/Sialyl Lewis a (SLe^a) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe^x and antibodies directed against related Lewis epitopes, Le^x and Le^a, had no significant effect on adhesion. Three antibodies directed against SLe^a differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe^a structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe^a present on individual proteins, suggesting that presence and right presentation of SLe^a epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe^a interaction. SLe^x and related epitopes, Le^x and Le^a, seem to have limited relevance for colon cancer cell recognition of E-selectin.     

There is No Correlation Between Function and Lymphokine Production of HBs-Antigen-Specific Human CD4+-Cloned T Cells

The question whether antigen-specific human CD4+ T cells can be classified on the basis of appropriate and fixed lymphokine production patterns and their corresponding functions still remains to be elucidated. We generated ten CD4+ T-cell clones specific for HBsAg from HBsAb-positive but HBsAg-negative individuals. Seven of these clones exhibited helper activity for HBsAb response, while the three other clones did not. Both helper- and non-helper-type T-cell clones produced interleukin 4 (IL-4) after antigenic stimulation. By stimulation with phytohaemagglutinin (PHA) plus phorbol myristate acetate (PMA), three of the seven helper-type clones produced interleukin 2 (IL-2) in addition to IL-4. However, the other four helper-type clones did not produce IL-2 by such stimulation, although they continued the production of IL-4. All non-helper-type T-cell clones produced a large amount of IL-2, and some of them completely became an IL-2 producer after certain stimulation. These results suggested that both helper- and non-helper-type CD4+ T-cell clones specific for HBsAg might have no strict pattern of lymphokine production as in the TH1/TH2 dichotomy of murine CD4+ T cells. The data also revealed that lymphokine-producing capacity of individual cloned T cells is changeable depending upon the sort of activation.     

Suppression of Cytolytic T-Lymphocyte Responses through Inactivation of Non-T Accessory Cells

The suppressive properties of nonspecific cytotoxic T lymphocyte (CTL) populations, derived from murine fetal calf serum (FCS)-precultured cells expanded in interleukin 2 (operationally defined as FCS-CM-expanded cells), were investigated on CTL responses generated by syngeneic alloreactive lymphoid cells. Our results suggest that the addition of FCS-CM-expanded cell populations can inhibit the CTL response by elimination of the bone marrow-derived macrophage (BM M phi) population used as non-T accessory cells. Indeed, in the culture conditions used, removal of IL-2 by the FCS-CM-expanded cells as well as a direct inactivating effect on the CTL precursors (CTL-P) could be excluded as a reason for inhibition. On the other hand, we were able to show that the BM M phi population was very sensitive to the cytolytic activity exhibited by the inhibiting cells in a 3 h 51Cr-release assay and that the suppressor effect observed could be partially circumvented by a second addition of BM M phi on the second day after the initiation of the culture.     

Down-Regulation of TLR9 Expression Affects the Maturation and Function of Murine Bone Marrow-Derived Dendritic Cells Induced by CpG

Toll-like receptor 9 （TLR9） is expressed intracellularly by dendritic cells （DCs） and specifically recognizes unmethylated CpG motif. Recognition of TLR9 to CpG DNA can induce DC maturation followed by the subsequent immune responses. Here, RNA interference （RNAi） was used to identify the effect of CpG DNA signaling on DC function. The results showed that transfection of DCs with siRNA specific for TLR9 gene significantly down-regulated TLR9 expression. Immature DCs transfected with TLR9 siRNA did not differentiate into mature DCs with exposure to CpG. TLR9 siRNA-treated DCs expressed low levels of MHC II and CD40 without reducing endocytosis. Furthermore, TLR9 siRNA-transfected DCs exhibited a decreased allostimulatory capacity in a lymphocyte proliferation assay and attenuated Thl responses by decreasing IL-12p70 production. Our findings indicate that siRNA in silencing TLR9 gene in DCs may offer a potential tool to study the TLR9-CpG pathway.     

Inhibition of Neutrophil‐Dependent Cytotoxicity for Human Endothelial Cells by ACE Inhibitors

Angiotensin‐converting enzyme inhibitors (ACEi) have immunomodulating properties and have been suggested to protect against endothelial injury, for example myocardial infarction and reperfusion injury. We tested whether two ACEi (captopril and enalapril), differing in a thiol group, protected human umbilical vein endothelial cells (HUVEC) from cytotoxicity induced by polymorphonuclear neutrophils (PMN) in vitro, when cells were activated by tumour necrosis factor‐α (TNFα) or the arachidonate derivative lipoxin‐A₄ (LXA₄), using separate cytotoxicity pathways. When ⁵¹Cr labelled HUVEC were treated with captopril (0–500 μm ) or enalapril (0–100 μm ) for 2 h and then activated by TNFα (100 ng/ml) for 24 h, a significant, dose‐dependent reduction of ⁵¹Cr release was observed. Similarly, captopril reduced ⁵¹Cr release when LXA₄ (0.1 μm ) was used to stimulate PMN for 4 h. Among previously defined mechanisms of significance for the cytotoxic reaction, expression of ICAM‐1, but not intracellular Ca²⁺ changes in PMN or PMN adherence to HUVEC, were reduced by ACEi treatment. Moreover, both ACEi inhibited HUVEC surface expression of TNFα receptor I (but not II). Thus, these ACEi, particularly captopril, interfere with PMN‐induced cytotoxicity for endothelial cells by modulating pro‐inflammatory surface receptors, which is a novel effect that might be explored for further therapeutic approaches.     

Protopine Inhibits Heterotypic Cell Adhesion in MDA-MB-231 Cells Through Down-Regulation of Multi-Adhesive Factors

Background

A Chinese herb Corydalis yanhusuo W.T. Wang that showed anticancer and anti-angiogenesis effects in our previous studies was presented for further studies. In the present study, we studied the anticancer proliferation and adhesion effects of five alkaloids which were isolated from Corydalis yanhusuo.

Materials and Methods

MTT dose response curves, cell migration assay, cell invasion assay, as well as three types of cell adhesive assay were performed on MDA-MB-231 human breast cancer cells. The mechanism of the compounds on inhibiting heterotypic cell adhesion were further explored by determining the expression of epidermal growth factor receptor (EGFR), Intercellular adhesion molecule 1 (ICAM-1), αv-integrin, β₁-integrin and β₅-integrin by western blotting assay.

Results

In five tested alkaloids, only protopine exhibited anti-adhesive and anti-invasion effects in MDA-MB-231 cells, which contributed to the anti-metastasis effect of Corydalis yanhusuo. The results showed that after treatment with protopine for 90 min, the expression of EGFR, ICAM-1, αv-integrin, β₁-integrin and β₅-integrin were remarkably reduced.

Conclusion

The present results suggest that protopine seems to inhibit the heterotypic cell adhesion between MDA-MB-231 cells, and human umbilical vein endothelial cells by changing the expression of adhesive factors.     

Epidermal Langerhans Cells as Accessory Cells in Con A Stimulation of T Lymphocytes in the Guinea-Pig

Stimulation of guinea-pig T cells by concanavalin A (Con A) requires Ia-antigen expressing accessory cells. Such functional accessory cells were identified among the normal epidermal cells and could be fractionated and enriched by centrifugation on discontinuous Percoll density gradients. Epidermal cells collecting at a density of 1.08 g/ml were the most effective in mediating Con A-dependent T cell proliferation, induced the highest activity of T cell growth factors (TCGF) and were enriched fivefold for Ia antigen expressing cells. A monolayer immunosorbent technique yielded a 35-fold enrichment of Ia antigen expressing epidermal cells. This cell fraction induced high levels of TCGF in the culture supernatants. Since the only cells in the normal epidermis expressing Ia antigens are the Langerhans cells, we conclude that the Langerhans cells may act as accessory cells for Con A stimulation of T cells.     

The Induction of Suppressor T Cells by Concanavalin A is Independent of Cellular Proliferation and Protein Synthesis

Factors that govern the induction of suppressor T cells after stimulation with concanavalin A (Con A) were investigated in a two-stage culture system. Normal mouse spleen cells were incubated with Con A in the presence of a variety of drugs and then assayed for suppressive activity by means of a secondary anti-sheep erythrocyte response in vitro. The inclusion of inhibitors of mitosis (vinblastine sulphate or mytomycin C) or protein synthesis (cycloheximide or pactamycin) into normal spleen cell cultures containing Con A failed to inhibit the subsequent development of suppressor cells. Furthermore, spleen cells from mice previously irradiated with 900 rad or injected with cyclophosphamide expressed a level of suppressor activity after Con A stimulation which was equivalent to that of normal spleen cells. However, the inclusion of drugs that inhibit microtubule or microfilament function (colchicine or cytocholasin B) did prevent suppressor cell induction. Kinetic studies also revealed that significant suppressor activity was detectable in normal spleen cells after only 3 h exposure to Con A. These results indicate that the induction of suppressor T cells in this system is a maturation event involving changes in the cell membrane and is entirely independent of protein synthesis and cellular proliferation.     

Antigen Presenting Cells in Human Decidual Tissue: III. Role of Accessory Cells in the Activation of Suppressor Cells

ABSTRACT: Human decidual antigen presenting cells (DAPCs) exposed to fetal cells in vitro induce generation of suppressor T cells among a population of peripheral blood lymphocytes. Human lymphocyte antigen (HLA)-class II positive antigen presenting cells were isolated from early normal human decidual tissue and from peripheral blood (PAPCs) by adhering Ficoll-Pa-que separated cell suspensions to fibronectin. In contrast to PAPCs, DAPCs pulsed with fetal antigens induced a radio-sensitive, Leu 1,2-positive T suppressor cell population. A nylon wool adherent B cell population is required during the in vitro induction of the suppressor cells. These suppressor cells impair primary mitogen and mixed lymphocyte culture (MLC) responses, generation of anti-trinitrophe-nyl (TNP) cytotoxic T lymphocytes, and antibody response of autologous and allogeneic lymphocytes. Only intact viable embryonic cells can effectively confer upon DAPCs the ability to induce T suppressor cells. The T suppressor cell induction by DAPCs primed with fetal antigens is restricted by the major histocompatibility complex. Our results show that the HLA-DR molecules are the most prominent restriction elements.