Melatonin inhibits granulocyte adhesion to ICAM via MT3/QR2 and MT2 receptors.

Neutrophils are cells of the innate immune system that first respond and arrive to the site of infection. Melatonin modulates acute inflammatory responses by interfering with leukocyte recruitment. It is known that melatonin modulates granulocyte migration though the endothelial layer thereby acting on the endothelial cell. Here we investigated whether melatonin could modulate granulocyte infiltration by acting directly on granulocytes. Granulocyte infiltration into the peritoneal cavity was investigated in mice kept at normal light/dark conditions and mice kept under constant lighting. To induce migration of neutrophils from the blood into the injury site via the endothelial layer, a bacterial product N-formyl-l-methionyl- l-leucyl- l-phenylalanine (fMLP) was injected into the peritoneal cavity. We found that the number of infiltrated granulocytes during the dark time was lower than that during the light time. It did not depend on circadian time. Moreover, the expression of an adhesion molecule, CD18, on granulocytes, was also lower during the dark time as compared with the light time. We have found that melatonin inhibited fMLP-induced CD18 up-regulation. Importantly, melatonin also inhibited the integrin-mediated granulocyte adhesion to intercellular adhesion molecule-coated plates. This study additionally showed that melatonin receptors MT2 and MT3/quinone reductase 2 (QR2) are expressed on granulocytes. Interestingly, melatonin increases the expression of its MT3/QR2 receptor. The fMLP-mediated CD18 up-regulation was inhibited by melatonin via MT2 receptor and the integrin-mediated granulocyte adhesion was inhibited by melatonin via MT3/QR2 and MT2 receptors. In conclusion, we show that melatonin suppresses granulocyte migration via endothelium by acting directly on granulocytes.

Regulation of T-cell-independent and T-cell-dependent antibody production by circadian rhythm and melatonin.

Melatonin is a hormone that has immunomodulatory activity and is believed to influence the production of antibodies in mammals. The aim of the present study was to investigate the effect of suppressed melatonin synthesis on the antibody production. BALB/c mice were immunized with T-cell-dependent (TD) and T-cell-independent (TI) antigens and kept under (i) normal lighting, (ii) constant exposure to light, (iii) exposed to light and treated daily with melatonin. It was revealed that melatonin modulated TD and TI antibody production. Suppressed melatonin synthesis increased the amount of IgM, IgG1, IgG2b and IgG3 antibodies after immunization with TI antigen. The level of TD antibodies IgM, IgG2a, IgG2b and IgG3 also increased, however, the antigen-specific antibodies of IgG1 isotype significantly decreased in mice exposed to light. Daily melatonin treatment brought the antibody level back to normal. The antibody concentration in the sera of mice kept at normal lighting was significantly higher when the immunizations were performed in the evening. The action of melatonin on B cells via MT2 receptor was shown in vitro and in vivo.

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation.

Plasma membrane damage and cell death during processes such as necroptosis and apoptosis result from cues originating intracellularly. However, death caused by pore-forming agents, like bacterial toxins or complement, is due to direct external injury to the plasma membrane. To prevent death, the plasma membrane has an intrinsic repair ability. Here, we found that repair triggered by pore-forming agents involved TMEM16F, a calcium-activated lipid scramblase also mutated in Scott's syndrome. Upon pore formation and the subsequent influx of intracellular calcium, TMEM16F induced rapid "lipid scrambling" in the plasma membrane. This response was accompanied by membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice exhibited compromised control of infection by Listeria monocytogenes associated with a greater sensitivity of neutrophils to the pore-forming Listeria toxin listeriolysin O (LLO). Thus, the lipid scramblase TMEM16F is critical for plasma membrane repair after injury by pore-forming agents.

Influence of circadian time and lighting conditions on expression of melatonin receptors 1 and 2 in murine lymphocytes.

AIM: To investigate the expression of membrane melatonin receptors in murine thymocytes, CD4(+) T-cells, bone marrow cells and B-cells according to melatonin concentration in the blood. MATERIALS AND METHODS: The levels of mRNA of melatonin receptors were investigated in the cells isolated during the day or during the night, corresponding to low and high melatonin concentrations in the blood. RESULTS: Low levels of Mtnr1 and Mtnr2 transcripts were detected in thymocytes and splenic B-cells. The expression of membranous melatonin receptors in B-cells corresponds to melatonin concentration in the blood. CONCLUSION: The expression of Mtnr1 and Mtnr2 in murine lymphocytes is very weak. Melatonin may be involved in regulation of the expression of Mtnr1 and Mtnr2 in murine B-cells.

The expression of MTNR3 and nuclear receptors in murine leucocytes.

AIM: The aim of this study was to investigate the expression of melatonin receptor MTNR3 and nuclear receptors in murine lymphocytes and their dependence on lighting conditions and circadian time. MATERIALS AND METHODS: The mRNA levels of melatonin receptors were investigated in cells isolated from thymus, spleen, lymph nodes and bone marrow during the day or during the night. RESULTS: The expression of MTNR3 in B-cells and bone marrow cells was much higher than in thymocytes and T-cells. Retinoic acid receptor-related orphan receptor A (Rora) was found mostly in thymocytes and cluster of differentiation 4 positive (Cd4(+)) T cells. Rorc was detected in thymocytes; its expression in peripheral T-cells was very low. Rorb was not detected in lymphocytes. MTNR3 transcripts in B-cells and Rorc transcripts in thymocytes increased during the day and decreased during the night. CONCLUSION: Circadian time and lighting could be involved in the regulation of the expression of melatonin receptors MTNR3 and Rorc.