Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes.

The sex hormones are known to affect innate immunity in humans. In this study we evaluated the immunomodulatory effects of testosterone in a model system comprising of all-trans retinoic acid differentiated HL-60 cells, and confirmed the results in human granulocytes and monocytes. Results showed that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest concentration of testosterone (120 µM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further demonstrated that the suppressive effect of testosterone has a short onset time. Our results suggest that testosterone affects the fast operating membrane bound androgen receptor or a rapid acting enzyme system. Testosterone, at pharmacological doses, was also shown to suppress generation of reactive oxygen species and interleukin-8 in human granulocytes and monocytes, respectively, to a similar extent as observed in differentiated HL-60 cells.

Direct interaction between CD91 and C1q.

C1q-mediated removal of immune complexes and apoptotic cells plays an important role in tissue homeostasis and the prevention of autoimmune conditions. It has been suggested that C1q mediates phagocytosis of apoptotic cells through a receptor complex assembled from CD91 (alpha-2- macroglobulin receptor, or low-density lipoprotein receptor-related protein) and calreticulin, with CD91 being the transmembrane part and calreticulin acting as the C1q-binding molecule. In the present study, we observe that C1q binds cells from a CD91 expressing monocytic cell line as well as monocytes from human blood. C1q binding to monocytes was shown to be correlated with CD91 expression and could be inhibited by the CD91 chaperone, receptor-associated protein. We also report data showing a direct interaction between CD91 and C1q. The interaction was investigated using various protein interaction assays. A direct interaction between purified C1q and CD91 was observed both by ELISA and a surface plasmon resonance assay, with either C1q or CD91 immobilized. The interaction showed characteristics of specificity because it was time-dependent, saturable and could be inhibited by known ligands of both CD91 and C1q. The results obtained show for the first time that CD91 recognizes C1q directly. On the basis of these findings, we propose that CD91 is a receptor for C1q and that this multifunctional scavenger receptor uses a subset of its ligand-binding sites for clearance of C1q and C1q bound material.

MCF-7 human mammary adenocarcinoma cells exhibit augmented responses to human insulin on a collagen IV surface.

Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell-based systems. We developed an insulin mitogenicity assay in MCF-7 human mammary adenocarcinoma cells, under low serum (0.1% FCS) and phenol red-free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10-fold dilution series, beta-estradiol was the most potent mitogen, followed by human IGF-1, human AspB10 insulin and native human insulin. AspB10 insulin was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were increased. Also, PI3K pathway activation by insulin was enhanced on a collagen IV surface. This study provided the first determination and ranking of the mitogenic potencies of standard reference compounds in an optimized MCF-7 assay. The optimized MCF-7 assay described here is of relevance for in vitro toxicological testing and carcinogenicity safety assessment of new insulin compounds.

The effect of dietary fish oil-supplementation to healthy young men on oxidative burst measured by whole blood chemiluminescence.

Dietary long-chain n-3 PUFA (n-3 LCPUFA) are thought to have immune-modulating effects, but the specific effects and mechanisms are not fully elucidated. The aim of this study was to determine whether dietary n-3 LCPUFA could affect ex vivo oxidative burst in healthy young men. The study had a randomised 2 x 2-factorial design in which subjects were randomly assigned to 8-week supplementation with capsules containing fish oil (about 2.9 g n-3 LCPUFA/d) or olive oil (control). Subjects were also randomly assigned to household use of oils and fat spreads with a high or a low 18:2n-6 content. At baseline and at the end of the intervention, the fatty acid composition of peripheral blood mononuclear cells (PBMC) was analysed by GLC and oxidative burst was studied in whole blood stimulated with zymosan using luminol-enhanced chemiluminescence. The PBMC content of n-3 LCPUFA was markedly increased by the fish oil-supplementation (P < 0.001, compared to the olive oil groups). No effect of the intervention was observed on neutrophil count, but one measure of the zymosan-induced oxidative burst was higher in the fish oil groups (P = 0.03) compared to the olive oil groups. The fat intervention did not in itself affect oxidative burst neither did it change the effect of the fish-oil intervention. The measures of oxidative burst at the end of the intervention period were found to be associated with the DHA content of PBMC (r 0.44, P = 0.016), suggesting a dose-response relationship. These results indicate that n-3 LCPUFA may have immuno-stimulating effects.

Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances.

In this paper we describe a new pyrogen assay using the human leukemia cell line HL-60. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to S. typhimurium but displays good sensitivity towards B. subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts C. albicans and S. cerevisiae is considerably better than obtained in other in vitro cell systems. Overall these results indicate that the HL-60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine.

Dry and moist heat sterilisation cannot inactivate pyrogenicity of Gram positive microorganisms.

In the monocytic cell line Mono Mac 6 pyrogens induce interleukin-6 secretion dose dependently. The aim of this study is to examine the interleukin-6 inducing capacity of Gram positive Staphylococcus aureus and Bacillus subtilis endospores after moist and dry heat sterilisation. Moist heat sterilisation of B. subtilis endospores for 15 min at 121 degrees C and 134 degrees C can only reduce the interleukin-6 inducing capacity to 57% and 63%, respectively, compared to untreated. Moist heat sterilisation of S. aureus for 60 min at 121 degrees C and 134 degrees C does not reduce the interleukin-6 inducing capacity of S. aureus. On the contrary moist heat sterilisation at 134 degrees C for 10, 20 and 40 min significantly increases the interleukin-6 inducing capacity compared to untreated S. aureus. This is confirmed in the rabbit pyrogen test. Dry heat sterilisation of B. subtilis endospores at 220 degrees C for 45 min reduces the interleukin-6 inducing capacity to 2% compared to untreated endospores. Dry heat treatment of S. aureus at 220 degrees C for 30 min only reduces the activity to 55%. However, after 250 degrees C for 30 min or 220 degrees C for 6h there is no detectable activity of S. aureus. In conclusion, neither the interleukin-6 inducing activity nor the pyrogenicity of S. aureus and endospores of B. subtilis can be inactivated by the heat sterilisation procedures described by the European Pharmacopoeia.

Regulation of interleukin-6 secretion in murine pituicytes.

Pituicytes, the astrocytic glial cells of the neural lobe, are known to secrete interleukin-6 and nitric oxide upon stimulation with various inflammatory mediators, i.e. interleukin-1beta. Nitric oxide is described to modulate the secretion of interleukin-6 in various cell types. The aim of the present study was to investigate the effect of nitric oxide on interleukin-1beta induced interleukin-6 secretion. Furthermore the effect of interferon-gamma on interleukin-6 and nitric oxide release was investigated. Cultures of pituicytes were prepared of neural lobes from male mice. The effect of interleukin-1beta and interferon-gamma on interleukin-6 and nitric oxide secretion was investigated in pituicytes cultured for 14 days. The secretion of interleukin-6 and nitric oxide was determined after 24 h of stimulation. Pituicytes secrete interleukin-6 upon stimulation with interleukin-1beta dose dependently but did not induce any detectable nitric oxide release. Co-stimulation with interferon-gamma and interleukin-1beta induced a significant nitric oxide release. In addition interferon-gamma inhibits interleukin-1beta induced interleukin-6 secretion dose dependently. The observed effect of interferon-gamma on interleukin-6 secretion was not affected by the specific inducible nitric oxide synthase inhibitor 1400W (N-(3-[aminomethyl]benzyl)acetamidine). Furthermore interferon-gamma dose dependently inhibits unstimulated interleukin-6 secretion. Use of the nitric oxide releaser DETA/NO (2,2'-(hydroxynitrosohydrazono)bis-ethanimine) demonstrated that nitric oxide does not inhibit interleukin-1beta induced interleukin-6 secretion. These results demonstrated that nitric oxide has no influence on interleukin-1beta induced interleukin-6 secretion in cultured pituicytes. However the results are showing that interferon-gamma has an inhibitory effect on interleukin-6 secretion.

Baclofen influences lipopolysaccharide-mediated interleukin-6 release from murine pituicytes.

Pituicytes, the glial cells of the neurohypophysis, secrete interleukin-6 upon stimulation with various inflammatory mediators, i.e. lipopolysaccharide. Previous studies have identified several receptors on pituicytes. This study investigates the effect of GABA(B) receptor activation on interleukin-6 release from pituicytes. Cultured murine pituicytes were stimulated for 24 h with lipopolysaccharide (0.5 ng/ml) to give a significant interleukin-6 release compared to control. The interleukin-6 release was significantly potentiated by the GABA(B) receptor agonist (R)-4-amino-3-(4-chlorophenyl) butanoic acid (R-baclofen; 10, 100 or 500 microM). However, R-baclofen itself (10, 100 or 500 microM) did not stimulate the interleukin-6 secretion. Furthermore, the potent GABA(B) receptor antagonists 3-[[(3,4-Dichlorophenyl)methyl]amino]propyl]diethoxymethyl) phosphinic acid (CGP52432; 30 or 300 microM) and (RS)-3-Amino-2-(4-chlorophenyl)-2-hydroxypropyl-sulphonic acid (2-OH-saclofen; 10 or 100 microM) did not remove the effect of R-baclofen (100 microM). Gamma-amino butyric acid (GABA; 30 or 300 microM) did not alter the lipopolysaccharide-mediated interleukin-6 response. After 30 min, intracellular cyclic AMP (cAMP) was higher in cells stimulated with lipopolysaccharide compared to control, and R-baclofen significantly inhibited this increase in cAMP. Nevertheless, neither lipopolysaccharide nor R-baclofen had any effect on intracellular cAMP after 24 h of stimulation. The results suggest that the effect of R-baclofen on lipopolysaccharide-stimulated interleukin-6 secretion is independent of GABA(B) receptors.