Also, melatonin was found to restore impaired Th cell activity in mice immunodepressed by ageing or by cyclophosphamide treatment [10, 11]
July 12, 2022
Also, melatonin was found to restore impaired Th cell activity in mice immunodepressed by ageing or by cyclophosphamide treatment [10, 11]. The present study was undertaken to examine the role of melatonin in ovalbumin-specific T cells by evaluating the secretion of IL-2, IFN-, IL-4 and IgG isotypes. by lymphokines has also been studied in a polyclonal system using lipopolysaccharide (LPS). It was shown that IL-4 induces the Streptozotocin (Zanosar) secretion of IgG1 and IFN- enhances the output of IgG2a . Melatonin (N-acetyl-5 methoxytryptamine) is regarded as the major hormone of the pineal gland. It is also secreted by lymphocytes and plays an important role in the immune system [6, 7]. Melatonin belongs to the group of indolamines. Indolamines have been shown to modulate the cytotoxicity of natural killer (NK) cells, change antibody responses, inhibit the proliferation of lymphocytes activated by mitogen and the production of IFN- by human T cells [8, 9]. It has also been reported that administration of melatonin increases the antibody response to various antigens and restores the antibody production in mice immunodepressed by acute restrain stress or by corticosterone treatment. Also, melatonin was found to restore impaired Th cell activity in mice immunodepressed by ageing or by cyclophosphamide treatment [10, 11]. The present study was undertaken to examine the role of melatonin in ovalbumin-specific T cells by evaluating the secretion of IL-2, IFN-, IL-4 and IgG isotypes. Results described here suggest that melatonin possibly acts on Th2-type cells, as evidenced by predominant secretion of IL-4, IgG1 antibody, but not IL-2, IFN- and IgG2a subtype production. MATERIALS AND METHODS Animals Inbred female BALB/c mice 8C10 weeks aged were obtained from the Institute’s Animal House Facility. Drug, antigen, antibodies Melatonin (Morepen Laboratories, Parwanoo, India), biotinylated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3 and steptavidin-labelled horseradish peroxidase (HRP; Sera Labs, Crawley Down, UK), ovalbumin (OVA), 2,2-azino-bis (3-ethylbenzthiazoline-6-sulphonic Streptozotocin (Zanosar) acid)-diammonium salt (ABTS), streptavidinCHRP, hydrogen peroxide and tetramethylbenzidine were procured from Sigma (St Louis, MO). Recombinant murine IL-2, IFN-, IL-4, monoclonal anti-mouse IFN- and biotinylated polyclonal goat anti-mouse IFN- antibodies were purchased from Genzyme (Cambridge, MA). Streptozotocin (Zanosar) Antibodies to IL-4 (11B11) were purchased from Texstar (Dallas, TX). Anti-IL-2 MoAbs (cocktail of TIB222, HB8794 and CRL 1698) were used as a culture supernatant. Cell lines and hybridomas The cell lines and hybridomas used in this study, HT-2 (CRL-1841), TIB222 (PC61.5.3), CRL 1698 (7D4) and HB 8794 (S4B6) were procured from ATCC (Rockville, MD). Immunization protocol OVA (2 mg/ml) was dissolved in PBS (001 m, pH 72) and emulsified in Freund’s complete adjuvant (FCA). Emulsion (100 l) was then injected intraperitoneally in the groups of five female BALB/c mice. Control animals were injected with PBS. After 1 week, a booster dose of the antigen was repeated. Five days before bleeding, the animals were injected subcutaneously daily with Streptozotocin (Zanosar) melatonin (10, 20 and 50 mg/kg body wt of mice). The control animals were immunized intraperitoneally with 01 ml each of placebo (PBS) and ethanolCPBS. After 2 weeks, draining popliteal lymph node (LN) cells from each group were removed and pooled for T cell proliferation and blood was BMP5 drawn and sera used for the quantification of IL-2, IFN-, IL-4 and IgG isotypes. T cell proliferation LN cells (15 105/well) obtained from different groups of five mice (i.e. immunized with OVA and melatonin, 1, 10, 20 and 50 mg/kg body wt, OVACPBS, OVACethanol, PBS, etc.) were cultured in triplicate wells. The cells were challenged with 100 g/ml of OVA along with different concentrations of melatonin (1, 10, 20 and 50 g/ml). The cultures were incubated for 72 h at 37C/7% CO2. The cells were pulsed with 05 Ci 3H-thymidine for 16 h before harvesting by automatic cell harvester (Skatron, Lier, Norway). 3H-thymidine incorporation was measured by standard liquid scintillation counting. Results are expressed as mean ct/min of triplicate cultures. Lymphokine assay IL-2 and IL-4 were measured using HT-2 cells The blood from experimental and control animals was collected 24 h after the last melatonin injection. The sera were separated and levels of IL-2 and IL-4 were measured by their respective abilities to induce the proliferation of HT-2 cells as described earlier . Briefly, 1 104/well of HT-2 cells were cultured in 96-well microtitre plates made up of medium and various concentrations of serum obtained from the control and experimental animals. For the selective inhibition of IL-2 and IL-4 lymphokines, antibodies to IL-2.