Carbon-Fiber Microelectrode Amperometry Reveals Sickle-Cell-Induced Inflammation and Chronic Morphine Effects on Single Mast Cells

Abstract

Sickle cell disease, caused by a mutation of hemoglobin, is characterized by a complex pathophysiology including an important inflammatory component. Mast cells are tissue-resident leukocytes known to influence a range of immune functions in a variety of different ways, largely through the secretion of biologically active mediators from preformed granules. However, it is not understood how mast cells influence the inflammatory environment in sickle cell disease. A notable consequence of sickle cell disease is severe pain. Therefore, morphine is often used to treat this disease. Because mast cells express opioid receptors, it is pertinent to understand how chronic morphine exposure influences mast cell function and inflammation in sickle cell disease. Herein, carbon-fiber microelectrode amperometry (CFMA) was used to monitor the secretion of immunoactive mediators from single mast cells. CFMA enabled the detection and quantification of discrete exocytotic events from single mast cells. Mast cells from two transgenic mouse models expressing human sickle hemoglobin (hBERK1 and BERK) and a control mouse expressing normal human hemoglobin (HbA-BERK) were monitored using CFMA to explore the impact of sickle cell-induced inflammation and chronic morphine exposure on mast cell function. This work, utilizing the unique mechanistic perspective provided by CFMA, describes how mast cell function is significantly altered in hBERK1 and BERK mice, including decreased serotonin released compared to HbA-BERK controls. Furthermore, morphine was shown to significantly increase the serotonin released from HbA-BERK mast cells and demonstrated the capacity to reverse the observed sickle cell-induced changes in mast cell function.

Single-cell analysis of mast cell degranulation induced by airway smooth muscle-secreted chemokines

“…MCs were isolated by peritoneal lavage and cultured as described previously. [23,26] The peritoneal cavity was injected with 8–10 ml of DMEM high glucose media supplemented with 10% bovine calf serum and 1% penicillin/streptomycin (all from ThermoFisher Scientific, Pittsburgh, PA). Extracted lavage fluid was stored on ice for transport and the collected cells were washed via centrifugation at 400 x g for 7 minutes followed by re-suspension in warm, fresh cell culture media containing either 0.5 μg ml −1 anti-TNP IgE (BD Biosciences, San Jose, CA), or vehicle for control conditions.…”

“…[25,26,29] Briefly, cultured MCs (with or without anti-TNP IgE pre-incubation) were mounted on a temperature controlled Petri dish holder (Warner Instruments, Hamden, CT) and positioned on the stage of an inverted microscope (Nikon, Tokyo, Japan). Prior to measurement, a carbon-fiber microelectrode connected to a headstage and a pulled glass micropipette loaded with a stimulating substance were mounted on Burleigh PCS-5000 piezoelectric micromanipulators (Olympus America, Center Valley, PA) to permit fine positional control in three dimensions.…”

“…Characterization of individual current spikes allows quantification of MC secretory function. [25,26,28] In this study, five key parameters were monitored to quantify mast cell secretory function: total serotonin per MC, spike area (Q), spike frequency, spike half-width (t 1/2 ), and spike rise time (t rise ). Total serotonin, calculated using the number of spikes per cell and the average amount of serotonin released per granule, reflects the overall degree of intragranular contents released per MC.…”

Electrochemical Analysis of Neurotransmitters

“…The recently discovered presence of post–spike plateau currents is indicative of partial-fusion, or kiss and run, events (11). Amperometric measurements have been applied to a variety of cell types including adrenal chromaffin cells (12), pheochromocytoma (PC12) cells (13), mast cells (14), and neurons (15, 16) to probe the pharmacology and biophysics of vesicular release events.…”

Metabolomic Profiling of Plasma and Erythrocytes in Sickle Mice Points to Altered Nociceptive Pathways

“…Serotonin modulates the descending fibers from the midbrain to the dorsal horn that inhibit the transmission of the painful stimuli. In mast cells from mouse models expressing human sickle hemoglobin, there was evidence of decreased serotonin released under chronic morphine exposure [31]. Sickle hyperalgia was shown to be modulated by the descending serotonergic anti-nociceptive system in HbSS-BERK (sickle) mice, while the serotonin concentration in the spinal cord dorsal is influenced by diet and correlated with hyperalgia [32].…”