Femtograms of Interferon-γ Suffice to Modulate the Behavior of Jurkat Cells: A New Light in Immunomodulation.

Since interferon-γ (IFN-γ) tunes both innate and adaptive immune systems, it was expected to enter clinical practice as an immunomodulatory drug. However, the use of IFN-γ has been limited by its dose-dependent side effects. Low-dose medicine, which is emerging as a novel strategy to treat diseases, might circumvent this restriction. Several clinical studies have proved the efficacy of therapies with a low dose of cytokines subjected to kinetic activation, while no in vitro data are available. To fill this gap, we investigated whether low concentrations, in the femtogram range, of kinetically activated IFN-γ modulate the behavior of Jurkat cells, a widely used experimental model that has importantly contributed to the present knowledge about T cell signaling. In parallel, IFN-γ in the nanogram range was used and shown to activate Signal transducer and activator of transcription (STAT)-1 and then to induce suppressor of cytokine signaling-1 (SOCS-1), which inhibits downstream signaling. When added together, femtograms of IFN-γ interfere with the transduction cascade activated by nanograms of IFN-γ by prolonging the activation of STAT-1 through the downregulation of SOCS-1. We conclude that femtograms of IFN-γ exert an immunomodulatory action in Jurkat cells.

Regulation of two rat mas-related genes in a model of neuropathic pain.

The mas-related gene (Mrg) family is a large family of G-protein-coupled receptors which are variable in number depending on species. The so-called sensory-neuron-specific receptors (SNSRs) make up a subset of the Mrg family, and several of these have been implicated in nociceptive processes. To verify their specific localization in sensory ganglia, we have determined the expression patterns of two of them, rMrgA and rMrgC, in a panel of rat tissues. The quantitative PCR results in the rat tissue panel indicate that, while several non-neuronal tissues contain significant levels of mRNA for both receptors, these two receptors are most highly expressed in dorsal root ganglia and trigeminal ganglia. Given this, we have examined the effects of spinal nerve ligation (SNL) on the expression of these genes. Peripheral neuropathy induced by ligation of spinal nerves at L5 and L6 resulted in a pronounced mechanical allodynia. These behavioral changes in tactile sensitivity were accompanied by significant decreases (10- to 100-fold) in the mRNA expression of both rMrgA and rMrgC exclusively in the L5 and L6 dorsal root ganglia ipsilateral to the SNL. In situ hybridization studies demonstrated that this decrease did not result from neuronal loss but rather from a reduction in the hybridization signals for rMrgC over small-to-medium diameter L5 and L6 dorsal root ganglia neurons. While the functional implications of the altered regulation of rMrgA and rMrgC in neuropathic pain models remain unclear, the results suggest that therapeutics targeting these receptors may have limited utility.

Endogenous and exogenous melanocortin antagonists induce anti-allodynic effects in a model of rat neuropathic pain.

A number of studies suggest melanocortin (MC) system involvement in nociceptive modulation. Although the mechanism through which this occurs is still unknown, experimental evidence would suggest a primary role of MC4 receptors. To further investigate the implication of this MC receptor subtype in chronic pain, we have studied the effects of several MC antagonists on spinal nerve ligation-induced nociceptive behavior in rats. The intrathecal injection of synthetic antagonists with different selectivity to MC4 receptor and of an endogenous antagonist (Agouti related protein; AgRP) reduced mechanical allodynia in neuropathic rats, as measured by von Frey hair test. Treatments produced an anti-allodynic effect at the dose of 1.5 nmol (25-30% maximum possible effect, MPE, P<0.05). To further investigate the possible physiological role of AgRP in pain modulation we studied its expression in both sham and neuropathic rat spinal cord and dorsal root ganglia (DRG) by quantitative real time PCR and immunohistochemistry. AgRP was present in both spinal cord and DRG, and its expression, was unchanged in neuropathic animals. In conclusion MC4 receptor antagonists with different selectivity profile, induce anti-allodynic effects in one of the most relevant neuropathic pain model. In addition the expression of AgRP in spinal cord and DRG suggests an endogenous tonic inhibitory control on MC system activity. In pathological conditions this steady control could be insufficient to cope with an over activated MC system leading to increase in nociception. These data suggest that targeting MC4 with synthetic antagonists could restore the balance and hence reduce nociception.

Gene expression profiling of melanocortin system in neuropathic rats supports a role in nociception.

The melanocortin (MC) system is involved in several biological functions. Its possible role in nociception has recently attracted attention in the field. Published data suggest that melanocortin antagonists are analgesic and agonists are hyperalgesic. Gene expression information about the MC system components (receptor, agonist and antagonist) in pain relevant areas is at present limited. To deepen our knowledge, we studied the expression of MC system components in nai;ve, sham and neuropathic rat spinal cord and dorsal root ganglia (DRG) by PCR and quantitative real-time PCR. MC4 receptor, proopiomelanocortin (POMC) and agouti-related protein (AgRP) transcripts were detected in both spinal cord and DRG, whereas MC3 receptor was detected only in the spinal cord. To study the relationship between the MC system and chronic pain, we used the chronic constriction injury model and gene expression analysis was performed in rats showing both tactile allodynia and thermal hyperalgesia. MC4 and POMC transcript were upregulated in the spinal cord of neuropathic rats, whereas MC3 and AgRP expression were unaffected. Thus, this study demonstrates for the first time the presence of AgRP in the spinal cord and DRG, suggesting that it could play a role in the regulation of MC system activity. In addition, the upregulation of POMC and MC4, in parallel with the presence of tactile allodynia and thermal hyperalgesia, further supports the idea of MC system involvement in nociception.

Expression of fractalkine and its receptor, CX3CR1, in response to ischaemia-reperfusion brain injury in the rat.

Fractalkine is a neuronally expressed chemokine that acts through its G-protein-coupled receptor CX3CR1, localized on microglial and immune cells. Fractalkine might be involved in neuroinflammatory processes secondary to neuronal damage, which normally occur in a time frame of days after ischaemia. We evaluated by in situ hybridization and immunohistochemistry the expression of fractalkine and CX3CR1 in the rat brain, after a transient occlusion of the middle cerebral artery. We found that at 12 h after ischaemia neuronal fractalkine expression was transiently increased in scattered necrotic neurons of the cortex and lost from the ischaemic striatum. At 24 and 48 h after ischaemia, fractalkine immunoreactivity was strongly increased in morphologically intact cortical neurons of the ischaemic penumbra where also the stress-inducible HSP-72 was strongly up-regulated. The intensity of fractalkine immunoreactivity of neurons in the penumbra returned to basal levels at 7 days after ischaemia. Fractalkine synthesis was also induced in endothelial cells of the infarcted area, at 48 h and 7 days after ischaemia. CX3CR1 expression was detected in the activated microglial cells of the ischaemic tissue 24 and 48 h after ischaemia, and became strongly up-regulated in macrophages/phagocytic microglia inside the infarcted tissue 7 days after ischaemia. These data suggest that fractalkine may participate in the activation and chemoattraction of microglia into the infarcted tissue, and contribute to the control of leucocyte trafficking from blood vessels into the injured area.

Flow cytometric analysis of inflammatory cells in ischemic rat brain.

BACKGROUND AND PURPOSE: Inflammation plays a key role in cerebral ischemia through activation of microglia and infiltration by leukocytes. Flow cytometry is a well-established method for quantitative and qualitative analysis of inflammatory cells. However, this technique has not been applied to the study of cerebral ischemia inflammation. The aim of this study was to establish a flow cytometric method to measure inflammatory cells in ischemic brain. METHODS: To perform flow cytometry on brain tissue, we developed 2 cell-isolation methods based on different mechanical dissociation and Percoll gradient separation techniques. The methods were tested on a rat model of permanent middle cerebral artery occlusion. Morphological and immunophenotypic analyses, with the use of anti-CD11b, anti-CD45, and alphabeta T-cell receptor antibodies, were employed to identify and quantify inflammatory cells. RESULTS: Both methods gave consistent results in terms of yield and reproducibility. The cell suspension contained granulocytes, macrophages, lymphocytes, and neural cells. Morphological and immunophenotypic analyses enabled the identification of a cell-scatter gate (R1a) enriched in inflammatory cells. With both methods, a higher number of events in R1a were recorded in the ischemic hemisphere than in the nonischemic hemisphere (P< or =0.001). CD11b, CD45, and alphabeta T-cell receptor staining confirmed that this augmentation was a reflection of the increase in the number of granulocytes, cells of the monocytic lineage, and lymphocytes. CONCLUSIONS: Quantitative flow cytometric analysis of ischemic rat brain is feasible and provides a reliable and rapid assay to assess neuroinflammation in experimental models of brain ischemia.