Modulation of pain by immune factors : a study on interferon-g and its receptor in nociceptive transmission

Abstract

Immune cytokines have been implicated as important neuromodulators, contributing to neuronal plasticity both in the adult and developing nervous system. In addition, they have been assigned a potential role in the generation of pathological pain, which may occur as a severe symptom following disease states in the nervous system. The underlying cellular mechanisms by which cytokines contribute to such pain have yet to be explored. A region of importance for the generation and maintenance of pathological pain is the dorsal horn of the spinal cord, where neuronal hyperexcitability, i.e. central sensitisation, may develop.

The present thesis project was undertaken with the aim of elucidating how the cytokine interferon-gamma (IFN-gamma) may modulate the transmission of nociceptive information in the dorsal horn of the rat spinal cord. In order to obtain a first indication of how IFN-gamma may exert its action, the cellular localisation of the receptor for IFN-gamma (IFN-gammaR was examined at the ultrastructural level in the dorsal hom. The results showed that immunoreactivity for the IFN-gammaR was primarily concentrated in postsynaptic densities in the superficial layers (Rexed's laminae 1 and 11) of the dorsal hom, which indicates that IFN-gamma may influence the transmission of nociceptive information at the synaptic level. Ligation of dorsal roots revealed a minor transport of receptors from the dorsal root ganglia (DRG) to the dorsal horn, which was also confirmed by the detection of scattered immunopositive presynaptic terminals, mainly in laminal 1.

Initial experiments on the effect of IFN-gamma on synaptic transmission were performed in primary cultures of hippocampal neurones. Whole cell patch clamp recordings revealed that the cytokine had no acute effects (secmin), but, on the other hand, mediated a significant increase in a-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) receptor-mediated spontaneous excitatory activity, as recorded after 48 h. This enhancement was not correlated to any changes in AMPA receptor (AMPAR) distribution in dendrites, determined by quantitative immunohistochemistry of the AMPAR subunit Glur1, or differences in AMPAR protein or mRNA levels. Prolonging the IFN-gamma-treatment to two weeks, however, resulted in a reduction in GluR1 density at postsynaptic sites, most likely in an activity-dependent manner, and furthermore, in normalised spontaneous activity frequencies. A significant reduction in GluR1, following prolonged IFN-gamma-treatment, was also observed in studies of cultured dorsal horn neurones. This decrease was blocked by concurrent treatment with the nitric oxide synthase (NOS) inhibitor, L-NG-mono-methyl-arginine (L-NMMA). In contrast to hippocampal neurones, the dorsal hom neurones exhibited significant increases in both frequency and amplitude of spontaneous excitatory activity following two weeks of IFN-gamma-treatment. These results may be explained by the fact that GluR1 is primarily localised to inhibitory interneurones in the spinal dorsal hom whereas to excitatory neurones in the hippocampus.

In order to evaluate how IFN-gamma-treatment may modulate dorsal horn neuronal responsiveness to primary afferent input, we developed a two-compartment in vitro model in which embryonic DRG and dorsal horn neurones are cultured separately but are in synaptic contact with each other. The model allows selective and efficient stimulation of DRG axons, while postsynaptic events are recorded in the dorsal horn neuronal network. Application of low and high frequency stimulation to the DRG axons resulted in wind-up of action potentials and long-term potentiation-like activity, respectively, i.e. electrophysiological features associated with central sensitisation. The model, thus, provides a good basis for analysis of how the synaptic transmission between DRG and dorsal hom neurones may be modulated. IFN-gamma-treatment for two weeks of the dorsal hom neurones in the outer compartment resulted in markedly enhanced responsiveness to low frequency stimulation of DRG axons, which further indicates that the dorsal horn neuronal network is sensitised.

In conclusion, this thesis provides further insight into the potential role of an immune regulatory cytokine, IFN-gamma, in nociceptive transmission. The results show that IFN-gamma may induce central sensitisation by reducing inhibitory control, i.e. disinhibition, of dorsal horn neuronal network activity. This proposes a potential mechanism for how prolonged presence of cytokines during disease states in the nervous system may contribute to generation of pathological pain.