CFMA measurements were conducted on peritoneal mast cells isolated from HbA-BERK, hBERK1 and BERK mice following 3 weeks of morphine treatment (PBS was used for control conditions). Measurement from an individual cell produced a current trace consisting of a collection of current spikes each corresponding to an individual degranulation event. The focus of this work was to explore the role of mast cells in SCD and the influence of morphine on mast cell function, both alone and in the context of chronic inflammation as modeled by the hBERK1 and BERK transgenic mice. Single mast cells were stimulated locally with the calcium ionophore A23187, which was selected as a universal mast cell stimulant that would limit bias toward a specific activation pathway. For the purpose of fulfilling these aims, four characteristics of the CFMA traces, plotted as time versus current, were analyzed among the experimental conditions: spike area (Q), spike frequency, spike half-width (t1/2), and spike rise-time (trise) (). Each spike characteristic reports on a different element of the exocytosis process. Analyzing the perturbations in several spike characteristics between experimental conditions provides a unique description of the mechanisms regulating the observed change in mast cell function.
Spike area (Q), the integral of each individual current spike over time, is a measure of charge, and thus represents the number of electrons transferred per release event. Because the oxidation of serotonin is a two-electron process, the area of an individual spike can be converted to the number of serotonin molecules released per granule. Spike frequency is calculated as the number of release events detected over the total release time and corresponds to the efficiency of the overall granule transport, docking and fusion mechanisms. Together, total Q and spike frequency can be combined to reveal the amount of serotonin released per cell (taking into account that the microelectrode covers only ~10% of the cell surface area and assuming equal secretion from all regions of the cell). In addition to spike area and frequency, spike rise-time (trise
) and half-width (t1/2
) values are monitored as a measure of the serotonin release kinetics from each granule fusion event. Trise
is calculated as the time between 10% and 90% of the full spike height on the rising phase of each current spike. Trise
reflects the amount of serotonin not directly associated with the chondroitin sulfate biopolymer matrix. Upon fusion, this free’ serotonin diffuses to the electrode surface more rapidly than the bulk of the intragranular serotonin that interacts strongly with the negatively charged matrix. Trise
is heavily influenced by the dilation of the initially formed fusion pore (Between the granule and the plasma membrane) to the maximally fused state. T1/2
, the width of the spike at half its full height, is a measure of the rate by which the biopolymer matrix expands and unfolds, releasing the remaining matrix- associated serotonin. Together, t1/2
reflect the biophysical forces that determine the peak shape (Sharp leading edge followed by a slower decay) typical of exocytotic release events.(33
To establish the impact of SCD-associated inflammation on mast cell function, mast cells isolated from PBS-treated HbA-BERK controls were compared to those from both hBERK1 and BERK mice (). The hBERK1 and BERK conditions demonstrated 27% and 58% reductions in Q, respectively (). Although this effect was significant in only the BERK mouse, the observed decrease trends with increasing sickle Hb expression. In addition, both hBERK1 and BERK mast cells released their granular contents less efficiently, resulting in significantly decreased spike frequencies by 34% and 32%, respectively (). When considered in concert versus HbA-BERK controls over the course of a 30 second release, these two effects resulted in a modest, though not statistically significant, decrease in overall serotonin release of 37%(1.97×109 fewer molecules per cell) for hBERK1 mast cells and a greater, significant decrease of 72%(3.84×109 fewer molecules per cell) in the BERK condition. These data suggest that the chronic inflammation in SCD induces mast cells to release less serotonin per exocytotic event as a result of either decreased granule loading or decreased percent serotonin released per granule, as regulated by a reduction in secretion driving forces. The relative magnitude of this effect appears to be dependent on disease severity. The reduced frequency of individual release events observed in both hBERK1 and BERK mast cells likely result from a decrease in either granule trafficking or fusion efficiency. Granule trafficking effects often occur due to perturbations of the microtubule transport machinery, whereas fusion efficiency is affected by changes in membrane stability. Unlike the observed changes in Q, changes in spike frequency observed in hBERK1 and BERK mice appear to be independent of disease severity.
Figure 3 Representative current traces collected using CFMA to analyze degranulation behavior of mast cells isolated from HbA-BERK (A), hBERK1 (B), and BERK (C) mice in the absence of morphine treatment. Mast cells were stimulated locally 4 with a 3 s bolus of (more ...)
Figure 4 The effect of sickle Hb expression and the corresponding chronic inflammation on mast cell function was explored using CFMA. Mast cells from PBS-treated HbA-BERK (n=77), hBERK1 (n=28) and BERK (n=30) mast cells were analyzed by CFMA. Spike area (A), spike (more ...)
Considering the observed sickle Hb-induced decrease in the number of secreted serotonin molecules (as indicated by Q), it is expected that, in the absence of changed release kinetics, t1/2
would decrease because it should take less time to release a smaller amount of serotonin. However, mast cells from both hBERK1 and BERK mice demonstrated significantly larger t1/2
values than those from the control mice (). Similarly, trise
values increased by 79% and 39%, respectively for hBERK1 and BERK mast cells compared to HbA-BERK controls (). This effect is also counter to the expected decrease resulting from the smaller Q values observed for both hBERK1 and BERK mice, with hBERK1 demonstrating the greatest increases in both t1/2
, due to the smaller decreases in Q compared to BERK mast cells. It has been shown in other granulated cell types that individual exocytosis events do not release the full mediator content of each granule.(9
) Although serotonin loading effects cannot be ruled out entirely, the increase in t1/2
measured herein despite corresponding decreases in Q for both hBERK1 and BERK mast cells suggests a mechanism of decreased serotonin released per granule rather than a decrease in overall granule loading.
Together, the observed decreases in Q and spike frequency, in addition to the somewhat counterintuitive increases in both t1/2 and trise suggest that the chronic inflammation present in both hBERK1 and BERK mice modulates mast cell serotonin secretion through a multifaceted mechanism involving both the serotonin release efficiency as well as membrane driving forces that alter granule fusion. Although the decrease in serotonin released (as indicated by decreased Q values) appears to be controlled in part by both decreased rate of transition from fusion pore to ‘full’ fusion (as indicated by increased trise values) and slower biopolymer matrix unfolding (as indicated by decreased t1/2 values) rather than decreased serotonin storage, further research will be required to further clarify the mechanism of this process. Similarly, the frequency effects observed in hBERK1 and BERK mast cells may also result from the same decreased membrane driving forces. The combination of changes in Q, spike frequency, t1/2, and trise observed in mast cells from hBERK1 and BERK mice indicate the serotonin release process in SCD is modulated by multiple compounding mechanisms.
To explore the effect of chronic morphine treatment on mast cell function independent of the inflammation associated with SCD, the serotonin release dynamics of mast cells isolated from HbA-BERK mice treated with either morphine or PBS were analyzed. On average, mast cells from morphine-treated mice released 172% more serotonin per granule than those from PBS-treated controls with no significant change in frequency (). When the effects of Q and spike frequency are combined as a measure of total serotonin released, a 162% increase in overall serotonin released per cell is observed, corresponding to 8.66×109 more serotonin molecules, and is entirely due to increased serotonin released per granule. Interestingly, chronic morphine exposure resulted in a small, significant increase in t1/2 (19%), which is expected considering the large observed increase in Q (). This expected result reflects the inherent association between t1/2 and Q. Other explanations for this t1/2 increase require invocation of an unnecessarily complex regulatory mechanism. No morphine-induced increase in trise was observed for mast cells from HbA-BERK mice ().
Figure 5 The effect of chronic morphine (MS) treatment on mast cells in the absence of chronic inflammation was explored. Mast cells from HbA-BERK mice treated with either PBS (n=77) or morphine (n=47) were analyzed by CFMA. Spike area (A), spike frequency (B), (more ...)
Unlike the complex disease-induced change in mast cell function described above, the observed effect of morphine treatment on mast cells in HbA-BERK mice appears to originate from a simpler mechanism. The large morphine-induced increase in serotonin released per granule is not associated with changes in the monitored spike parameters other than Q. These data suggest morphine treatment induces mast cells to either store more serotonin per granule or release a greater portion of its granular contents per release event. The lack of unexpected changes in t1/2 or trise suggest that the driving forces of granule fusion are not markedly altered by chronic morphine exposure in mast cells from HbA-BERK mice. Therefore, increased serotonin loading is more likely responsible for the large increase in serotonin released per granule from these mast cells.
Finally, the effect of chronic morphine treatment on hBERK1- and BERK-derived mast cells was investigated. With respect to the effect on serotonin released per granule, mast cells from BERK mice demonstrated significantly increased Q values sufficient to more than recover the 58% reduction in Q attributed to the expression of sickle Hb (). Although statistically insignificant, a smaller recovery trend was also seen in hBERK1-derived mast cells (). Interestingly, although in HbA-BERK mice morphine had no effect on release frequency, mast cells from both hBERK1 and BERK mice responded to morphine treatment by reversing the depressed frequencies observed for the PBS conditions in each (). In addition, treatment with morphine significantly recovered the sickle Hb-induced increases in trise in mast cells from both hBERK1 and BERK mice (). A similarly significant recovery effect was observed in the t1/2 values in the hBERK1 condition (), and although a morphine-induced recovery of the t1/2 values was not observed for the BERK condition, this is attributable to the relatively smaller initial sickle Hb-induced effect in these mice ().
Figure 6 The effect of morphine on mast cell function in sickle cell mice was explored. Mast cells from MS-treated HbA-BERK (n=47), hBERK (n=52) and HbA-BERK (n=47) mice were compared to PBS-treated controls (n=77 for HbA-BERK, n=28 for hBERK, n=30 for BERK) and (more ...)
Given the large morphine-induced increase in Q in mast cells from HbA-BERK control mice, the observed recovery of Q in BERK mice, although important, is perhaps less surprising compared to the morphine-induced recoveries observed for spike frequency, trise, and t1/2 despite the lack of morphine-mediated effects on any of these parameters in the HbA-BERK mice (with the exception of t1/2 values in the HbA-BERK mice as mentioned above). For all measured parameters, it is worth noting that the morphine-induced recoveries that were observed did not grossly exceed the corresponding values measured from morphine treated HbA-BERK controls (). Interestingly, Q was the only measured parameter demonstrating overcompensation behavior in response to treatment with morphine (relative to mast cells from PBS-treated HbA-BERK mice). Nonetheless, Q values in morphine-treated hBERK1 or BERK mice did not exceed those from morphine-treated HbA-BERK mice (). It is likely that the increased amount of serotonin released from mast cells from morphine-treated hBERK1 and BERK mice (as demonstrated by an increase in Q) results from a combination of granule loading and increased granule fusion driving forces. Because granule loading was found to be the sole observed mechanism of morphine-mediated regulation of serotonin release in HbA-BERK mast cells, these data indicate that the morphine-induced increase in serotonin loading is independent of inflammation. In contrast, this data suggests the ability of morphine to recover mast cell functionality via regulation of matrix unfolding and membrane driving forces (the mechanisms likely responsible for decreasing the amount of serotonin released from mast cells in hBERK1 and BERK mice) as measured by spike frequency, trise and t1/2, may be limited to levels similar to morphine-treated controls. According to this hypothesis, these findings argue matrix unfolding effects and membrane driving forces are rate limited under normal conditions, resulting in minimal perturbation of these factors in non-SCD mice upon morphine exposure.
To summarize, this research proposes that chronic inflammation in mice expressing human sickle Hb impairs the secretion of serotonin from mast cells through multiple mechanisms, including fusion pore formation/modulated membrane driving forces and matrix expansion efficiency. Chronic morphine treatment was found to act differently on mast cells from non-sickle cell mice (HbA-BERK) than those expressing human sickle Hb (hBERK1 and BERK). In the absence of SCD-associated inflammation, morphine exposure induced mast cells to increase the amount of serotonin released per granule, a relatively simple mechanism likely resulting from increased serotonin loading. However, morphine treatment induced a more complex change in mast cells isolated from hBERK1 and BERK mice. Whereas only serotonin loading effects were observed in mast cells from HbA-BERK mice, morphine induced marked recovery of all the sickle cell-induced perturbations in mast cell function in both hBERK1 and BERK mice. Morphine was observed to both increase serotonin loading and restore membrane driving forces to levels similar to those of morphine-treated control mice. Given the capacity for mast cells to influence the inflammatory microenvironment and the importance of inflammation in the progression of SCD, these findings offer unique insight into 1) the significantly altered mast cell function in response to sickle-cell induced inflammation, 2) the large morphine-induced increase in serotonin released per mast cell, and 3) the capacity for morphine to compensate for sickle-cell induced changes in mast cell function.
Any broad-reaching implications of these findings will require significant additional research to characterize both the extent to which mast cells influence the chronic inflammation in SCD as well as the relative importance of morphine in regulating mast cell function when considering available treatment options. Furthermore, in light of tissue-specific mast cell heterogeneity, further work is required to evaluate the universality of these findings. However, it is clear from this study that mast cell function is indeed altered in mice expressing sickle Hb. It is apparent that the use of morphine to treat the pain associated with SCD may also influence the inflammatory state of the disease. Deciphering the root cause of sickle Hb-induced mast cell effects, and determining whether morphine complicates or improves the pathophysiology of SCD, and the extent of either, will be the subject of future collaborative research in this area.