Reciprocal modulation of helper Th1 and Th17 cells by the ß2-adrenergic receptor agonist drug terbutaline.

Catecholamine hormones are powerful regulators of the immune system produced by the sympathetic nervous system (SNS). They regulate the adaptive immune system by altering T-cell differentiation into T helper (Th) 1 and Th2 cell subsets, but the effect on Th17 cells is not known. Th17 cells, defined, in part, by chemokine receptor CCR6 and cytokine interleukin (IL)-17A, are crucial for mediating certain pathogen-specific responses and are linked with several autoimmune diseases. We demonstrated that a proportion of human Th17 cells express beta 2-adrenergic receptor (ß2AR), a G protein-coupled receptor that responds to catecholamines. Activation of peripheral blood mononuclear cells, which were obtained from venous blood drawn from healthy volunteers, with anti-cluster of differentiation 3 (CD3) and anti-CD28 and with a ß2-agonist drug, terbutaline (TERB), augmented IL-17A levels (P < 0.01) in the majority of samples. TERB reduced interferon gamma (IFNγ) indicating that IL-17A and IFNγ are reciprocally regulated. Similar reciprocal regulation was observed with dbcAMP. Proliferation of Th cells was monitored by carboxyfluorescein diacetate N-succinimidyl ester labeling and flow cytometry with antibody staining for CD3 and CD4. TERB increased proliferation by a small but significant margin (P < 0.001). Next, Th17 cells (CD4+ CXCR3- CCR6+ ) were purified using an immunomagnetic positive selection kit, which removes all other mononuclear cells. TERB increased IL-17A from purified Th17 cells, which argues that TERB acts directly on Th17 cells. Thus, hormone signals from the SNS maintain a balance of Th cells subtypes through the ß2AR.

A simple and rapid radioimmunoassay for human interleukin-6.

Studies were undertaken to develop a sensitive radioimmunoassay for human IL-6 using commercially available reagents. The assay utilized a polyclonal rabbit anti-IL-6 binding to iodinated IL-6; the reaction was carried out in solution and the immune complexes were precipitated using anti-rabbit IgG coupled to magnetic particles. Using a format where sample IL-6 was added in advance of the radiolabelled IL-6, the working range of the assay was found to fall between 0.1 and 10 ng/ml (IC50 around 1 ng/ml) for a 50 microliters sample volume, and was run overnight. However, the assay could be completed in 2 h, using a direct competitive format when less sensitivity is required (working range 1.5-25 ng/ml, IC50 12 ng/ml). The RIA correlated directly with a standard functional assay for IL-6 (proliferation of the mouse hybridoma B9.9) for both recombinant IL-6 and natural IL-6 from human monocytes. The total assay variance was less than 12% and no reactivity with interleukins-1-5 was found. Using the RIA, IL-6 produced in culture by human monocytes in response to various stimuli (LPS, IL-1, dibutyryl cAMP) was measured.

[Effect of substances that alter the level of intracellular cAMP on the reaction of human lymphocytes blast transformation]. / Vliianie veshchestv, izmeniaiushchikh uroven' vnutrikletochnogo tsAMF, na reaktsiiu blasttransformatsii limfotsitov cheloveka.

Effects of cholera toxin, cholera anatoxin, theophylline and dibutyryl cyclic AMP (db-cAMP) on the PHA-induced DNA synthesis have been studied. It was shown that toxin, db-cAMP and theophylline suppressed mitogen-induced 3H-TdR incorporation and blast-transformation in a dose-related manner. When both toxin and theophylline were given together to PHA-stimulated lymphocytes, the inhibitory effects of these two agents were additive. The inhibitory effects of db-cAMP and theophylline were additive too. This inhibitory effects could be due to the elevation of intracellular cAMP. Cholera anatoxin exerted an inhibitory action on the PHA-stimulated lymphocytes. The inhibitory effects on lymphocyte DNA synthesis by anatoxin and theophylline were not additive. These results suggest that the effects of cholera toxin are mediated by cyclic AMP, but effects of cholera anatoxin may be associated with some other mechanism.

Mechanisms of strain-dependent development of mast cells from mouse splenocytes.

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.

Lipopolysaccharide augments HLA-A,B,C molecule expression but inhibits interferon-gamma-induced HLA-DR molecule expression on cultured human endothelial cells.

The effect of bacterial lipopolysaccharide (LPS) on the expression of class I and II major histocompatibility complex (MHC) molecules on the surface of cultured human umbilical vein endothelial cells (HUVEC) was determined by indirect immunofluorescent staining followed by flow cytometric analysis. LPS at concentrations higher than 0.01 micrograms/ml augmented class I MHC (HLA-A,B,C) expression on HUVEC in a concentration-dependent manner. Optimal augmentation, approximately sixfold compared with control, was seen with 10 micrograms/ml of LPS. Time-course experiments indicated that the augmentation was maximal on Day 4. In contrast, LPS had no effect on the induction of class II MHC (HLA-DR) molecules and at concentrations higher than 0.01 micrograms/ml inhibited the interferon-gamma(IFN-gamma)-induced class II MHC expression. The inhibition was about 60% at the concentration of 100 micrograms/ml of LPS. Interleukin-1 (IL-1) had a similar effect as LPS on class I and II MHC expression. However, LPS appeared to affect MHC expression directly and not through production of IL-1 or cyclo-oxygenase pathway products, since anti-IL-1 antibodies or an inhibitor of cyclo-oxygenase pathway products, indomethacin, failed to reverse the effects of LPS. These data stress the role of LPS as a direct modulatory factor of class I and II MHC expression on endothelial cells during the development of immune and inflammatory response against Gram-negative bacteria.

Elevation of intracellular cyclic AMP induces an anergic-like state in Th1 clones.

Because elevated intracellular cAMP suppresses T cell receptor (TCR)-mediated effector activity and/or proliferation in response to antigen but does not always affect IL-2-stimulated proliferation, the effects of cAMP on a T lymphocyte response to antigen resemble antigen-induced anergy. To test the hypothesis that elevated cAMP induces anergy in T lymphocytes, we have precultured murine Th1 clones responsive to porcine myelin basic protein (PMBP) with dibutyryl cyclic AMP (dbcAMP) or forskolin and subsequently removed the dbcAMP or forskolin and measured the proliferative response of the clones to antigen and antigen-presenting cells (APC) in the presence or absence of exogenously added interleukin-2 (IL-2). Cells precultured with dbcAMP or forskolin for 3 days did not proliferate or produce IL-2 in response to antigen and APC, but did proliferate to antigen and APC in the presence of IL-2. Cells that had not been stimulated recently with antigen/APC or IL-2 were not affected by dbcAMP, while cells stimulated recently with antigen/APC and IL-2 were susceptible to the anergizing effect of dbcAMP. These observations support the hypothesis that elevation in intracellular cAMP in antigen-activated Th1 clones, prior to subsequent culture with antigen, induces a state of anergy.

Regulation of the in vitro early anamnestic antibody response by exogenous cholera enterotoxin and cyclic AMP.

Keyhole limpet hemocyanin was injected into the hind foot pads of rabbits. Six days later cell suspensions were prepared from the popliteal lymph nodes. Various amounts of hemocyanin (1 ng to 100 mug) were added to 1 times 10-7 cells to induce an anamnestic antibody response. Various amounts of cholera enterotoxin, which stimulates the enzyme adenylate cyclase, or dibutyryl cyclic adenosine 3'5'-monophosphate (AMP), were added to the cultures with or without hemocyanin. De novo synthesis of antibody, protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from radioactive precursors was assayed. The addition of cholera toxin or dibutyryl cyclic AMP for the first 24 hr with optimal (1 mug) or supraoptimal (100 mug) amounts of hemocyanin enhanced antibody synthesis by at least 100 to 200%. Addition of the toxin or dibutyryl cyclic AMP for the same period to cells minus hemocyanin or with suboptimal amounts (1 to 100 ng) of antigen failed to enhance antibody synthesis. Addition of these agents for 72 to 120 hr to hemocyanin-induced cultures consistently inhibited antibody synthesis. These agents slightly inhibited DNA and RNA synthesis. The increase in protein synthesis caused by the toxin or dibutyryl cyclic AMP was almost totally accounted for by the increase in antibody synthesis. Neither toxin nor cyclic nucleotide promoted the antibody response in the presence of antibody to rabbit thymus-derived lymphocytes; these lymphocytes as well as bursa-equivalent lymphocytes were required for potentiation of the response. Macrophages were not required either for induction of the anamnestic response or for enhancement of this synthesis by cyclic nucleotide or cholera toxin. Both IgM and IgG antibody synthesis were regulated by exogenous cholera toxin and dibutyryl cyclic AMP. A number of possible cellular mechanisms of regulation of the antibody response through the cyclic AMP pathway were discussed. These included the effects of modifications of this pathway on the activities of T lymphocytes early (0 to 24 hr) and B lymphocytes late (72 to 120 hr) in the response and on the apparent reversal of high zone tolerance.