ABSTRACT
1 University of Modena and Reggio Emilia, Department of Biomedical Sciences, via G Campi 287, 41 125, Modena, Italy
2 Institute of Psychiatry, King’s College London SE5-9NU, UK
In the past few decades it is becoming more and more evident that major depression (MD) is associated with a profound unbalance between the nervous-, the endocrine- and the immune (IS)- systems. This suggests the possibility that molecules that regulate the homeostasis of these systems may contribute to the onset and/or the maintenance of MD. For instance, some cytokines, important neuro-endocrine-immuno modulators, have been proposed to have a role in MD as supported by the following observations: 1. Circulating pro-inflammatory cytokine levels are often elevated in MD; 2. Individuals suffering from immune-related disorders with high level of circulating cytokines often experience psychiatric symptoms; 3. Acute experimental activation of IS with endotoxin or therapeutically used cytokines, such as interferon (IFN) alpha, may induce depressive symptoms that are responsive to the treatment with standard antidepressant therapy.
It has been proposed that cytokines may contribute to the etiology of MD by inducing indolamine 2,3-dioxygenase (IDO) expression. IDO catalyzes the initial rate-limiting step in tryptophan (TPR) degradation along the kynurenine pathway (KP). Kynurenine (KYN), the initial product of TPR degradation, is further catalysed into neurotoxic end-products (including the NMDA receptor agonist quinolinic acid- QUIN) through steps catalyzed by kynurenine 3-monooxygenase (KMO) and kynureninase (Kynu). However, KYN can also be catabolised by kynurenine aminotransferase (KAT), into kynurenic acid (KYNA), which is a NMDA receptor antagonist, thus a potentially neuroptotective agent. A role for a disturbance in the equilibrium between QUIN and KYNA producing an alteration in the neuroprotective–neurodegenerative balance in the brain of patients with chronic depression, has been proposed in the neurodegeneration hypothesis of depression.
However, despite the heuristic value of this hypothesis, the mechanism(s) through which the unbalance of KP may lead to neurodegeneration and thus may possibly contribute to depression, is far to be clear.
This prompted us to investigate the effects of Interleukin (IL) 1 beta, IL-18 and IFN alpha on the expression of the IDO, KMO, Kynu and KAT mRNAs in an in vitro model of human neurons: SH-SY5Y (human neuroblastoma) cells. Cells were incubated for 24 hours with either IL-1 beta (10 ng/ml), IL-18 (100 ng/ml) or IFN alpha (100 ng/ml). quantification of the kynurenergic enzyme mRNAs expression performed by using quantitative Real Time PCR. Our studies show that, albeit the effects on the expression of the kynurenergic enzymes were specific for the different cytokines tested, there was a general unbalance of the KP toward the synthesis of neurotoxic end-product. In particular IFN alpha strongly induced the IDO mRNA expression (more than 900 –fold) in SH-SY5Y cells when compared to control untreated cells.
Moreover, we evaluated the possible cytotoxic effects induced by IL-1 beta (10 ng/ml), IL-18 (100 ng/ml) and IFN alpha (100 ng/ml) exposure for 24, 48 or 72 hours in SH-SY5Y cells by using crystal violet assay to detect cell number. Our results demonstrated that the time of exposure to these cytokines may be critical in affecting human neuroblastoma cell number. Moreover, these data clearly enlighten the cytotoxic effects of IFN alpha in this in vitro model of human neurons.
Finally, to evaluate whether the effects induced by cytokines treatments (in particular IFNalpha) on cell number are mediated by an increase in apoptosis, we tested SH-SY5Y cells treated for 72 hours with the pro-inflammatory cytokines for apoptosis with the Hoechst 33258 staining. Our results demonstrated that IFN alpha induced apoptosis in human SH-SY5Y neuroblastoma cells.
Together our findings provide further information on the molecular pathways involved in cytokine-induced effects in the brain and add a piece to the puzzle of what and how these factors or pathways may contribute to the pathogenesis of MD.