One important mechanism of cross-regulation by opposing cytokines is inhibition of signal transduction, including inhibition of Janus kinase-STAT signaling by suppressors of cytokine signaling. We investigated whether IFN-γ, a major activator of macrophages, inhibited the activity of IL-10, an important deactivator. Preactivation of macrophages with IFN-γ inhibited two key anti-inflammatory functions of IL-10, the suppression of cytokine production and of MHC class II expression. Gene expression profiling showed that IFN-γ broadly suppressed the ability of IL-10 to induce or repress gene expression. Although IFN-γ induced expression of suppressor of cytokine signaling proteins, IL-10 signal transduction was not suppressed and IL-10 activation of Janus kinases and Stat3 was preserved. Instead, IFN-γ switched the balance of IL-10 STAT activation from Stat3 to Stat1, with concomitant activation of inflammatory gene expression. IL-10 activation of Stat1 required the simultaneous presence of IFN-γ. These results demonstrate that IFN-γ operates a switch that rapidly regulates STAT activation by IL-10 and alters macrophage responses to IL-10. Dynamic regulation of the activation of different STATs by the same cytokine provides a mechanism by which cells can integrate and balance signals delivered by opposing cytokines, and extends our understanding of cross-regulation by opposing cytokines to include reprogramming of signaling and alteration of function.
Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP-release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non-junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non-junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.
Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury.
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