Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as ‘find/eat-me’ signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.

Micropatterned substrates offer a unique possibility to define and control spatial organization of biological cells at the microscale, which greatly facilitates investigations of the cell-to-cell communication in vitro. Here, we developed a simple micropatterning strategy to resolve various spatiotemporal characteristics of intercellular calcium wave (ICW) communication among isolated BV-2 microglial cells. By using a single-ring assembly, we found that the direction of the initial transmitter secretion was strongly correlated with the site of the cell at which the mechanical stimulus triggering the ICWs was imposed. By using multiring assemblies, we observed that the response ratio of the same outmost cells 160 μm away from the center increased from 0% in the single-ring assembly to 9.6% in the four-ring assembly. This revealed that cells located in the interring acted as regenerative amplifiers for the ICWs generated by the central cell. By using a special oval-type micropattern, we found that calcium mobilization in lamellipodia of a fusiform BV-2 microglia cell occurred 2.9 times faster than that in the middle part of the cell, demonstrating a higher region-specific sensitivity of lamellipodia to the transmitter. Taken together, our micropatterning strategy opened up new experimental prospects to study ICWs and revealed novel spatiotemporal characteristics of ICW communication including stimulation site-dependent secretion, regenerative propagation, and region-specific cell sensitivity.

Ultraviolet blood irradiation has been used as a physical therapy to treat many nonspeci¯c diseases in clinics; however, the underlying mechanisms remain largely unclear. Neutrophils, thē rst line of host defense, play a crucial role in a variety of in°ammatory responses. In the present work, we investigated the e®ects of ultraviolet light A (UVA) on the immune functions of human neutrophils at the single-cell level by using an inverted°uorescence microscope. N-Formylmethionyl-leucyl-phenylalanine (FMLP), a classic physiological chemotactic peptide, was used to induce a series of immune responses in neutrophils in vitro. FMLP-induced calcium mobilization, migration, and phagocytosis in human neutrophils was signi¯cantly blocked after treatment with 365 nm UVA irradiation, demonstrating the immunosuppressive e®ects of UVA irradiation on neutrophils. Similar responses were also observed when the cells were pretreated with H 2 O 2 , a type of reactive oxygen species (ROS). Furthermore, UVA irradiation resulted in an increase in NAD(P)H, a member of host oxidative stress in cells. Taken together, our data indicate that

To perform various physiological functions, erythrocytes possess a unique biconcave shape provided by a special architecture of the membrane-skeleton system. In the present work, we use a simple irradiation method to treat human erythrocytes with 365 nm ultraviolet-A (UVA) light at the single-cell level in vitro. Depending on the irradiation dose, UVA show protection of the biconcave profile against the detrimental action of distilled water. This protective effect can also be confirmed for saponin that damages the membrane-skeleton by vesiculation and pore formation. Interestingly, at two irradiation doses of UVA pretreatment, erythrocytes still seem to exhibit cell viability as tested by trypan blue assay even if distilled water or saponin is added. The oxidants hydrogen peroxide and cumene hydroperoxide partly simulate the protective effects. Taken together, these results demonstrate that 365 nm UVA irradiation can protect the biconcave profile of human erythrocytes through membrane-skeleton enhancement associated with a production of oxidants.

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