Activated polymorphonuclear cells promote injury and excitability of dorsal root ganglia neurons

https://doi.org/10.1016/j.expneurol.2007.11.024Get rights and content

Abstract

Therapies aimed at depleting or blocking the migration of polymorphonuclear leukocytes (PMN or neutrophils) are partially successful in the treatment of neuroinflammatory conditions and in attenuating pain following peripheral nerve injury or subcutaneous inflammation. However, the functional effects of PMN on peripheral sensory neurons such as dorsal root ganglia (DRG) neurons are largely unknown. We hypothesized that PMN are detrimental to neuronal viability in culture and increase neuronal activity and excitability. We demonstrate that isolated peripheral PMN are initially in a relatively resting state but undergo internal oxidative burst and activation by an unknown mechanism within 10 min of co-culture with dissociated DRG cells. Co-culture for 24 h decreases neuronal count at a threshold < 0.4:1 PMN:DRG cell ratio and increases the number of injured and apoptotic neurons. Within 3 min of PMN addition, fluorometric calcium imaging reveals intracellular calcium transients in small size (< 25 μm diam) and large size (> 25 μm diam) neurons, as well as in capsaicin-sensitive neurons. Furthermore, small size isolectin B4-labeled neurons undergo hyperexcitability manifested as decreased current threshold and increased firing frequency. Although co-culture of PMN and DRG cells does not perfectly model neuroinflammatory conditions in vivo, these findings suggest that activated PMN can potentially aggravate neuronal injury and cause functional changes to peripheral sensory neurons. Distinguishing the beneficial from the detrimental effects of PMN on neurons may aid in the development of more effective drug therapies for neurological disorders involving neuroinflammation, including painful neuropathies.

Introduction

Neuronal damage is generally associated with recruitment of immune cells to the site of injury, thus resulting in neuroinflammation. During the acute phase of neuroinflammation, circulating polymorphonuclear leukocytes (PMN or neutrophils) are among the first leukocytes to detect inflammatory signals that trigger PMN rolling along blood vessels, firm adhesion and transendothelial migration into endoneurial space (Nathan, 2006). Subsequently, activated PMN release several mediators of cellular toxicity to kill invading pathogens. As such, activated PMN come into close proximity with neurons in a plethora of neurological conditions, including neurodegenerative (multiple sclerosis, Parkinson's, Alzheimer's) and neurotraumatic disorders (spinal cord injury, ischemia, painful neuropathy). Although the inflammatory response is a critical component of healing and repair, abundant evidence in rodent models suggest that limiting PMN migration can positively influence the outcome of neuronal injury (Ryu et al., 2007, Yamasaki et al., 1997, Prestigiacomo et al., 1999, Connolly et al., 1996, Huang et al., 1999, Taoka et al., 1997).

Blocking PMN migration mediated by cytokine-induced chemoattractant-1 can ameliorate the neurological outcome following stroke (Yamasaki et al., 1997). Neutropenia or deficiency in the intercellular adhesion molecule ICAM-1 that mediates PMN migration confers neuronal protection after experimental cerebral ischemia and stroke (Connolly et al., 1996, Huang et al., 1999, Prestigiacomo et al., 1999, see also Dirnagl et al., 1999, Lo et al., 2003). Administration of anti-P-selectin antibody, which disrupts interaction between activated PMN and endothelial cells, attenuates tissue injury and improves motor performance following spinal cord compression (Taoka et al., 1997).

Neuropathic pain is a neurological disorder caused by nerve lesion whereby patients face unsatisfactory treatment options (Dworkin et al., 2003). In different animal models of sciatic neuropathy, PMN accumulate at the site of nerve lesion (Perkins and Tracey, 2000), as well as proximally in lumbar dorsal root ganglia (DRG) (Morin et al., 2007). Notably, neuropathic pain is associated with hyperexcitability of lumbar DRG neurons (Amir et al., 2005, Djouhri et al., 2006, Devor, 1999). Interestingly, neuropathic pain is attenuated by blocking PMN migration after 5-hydoxytryptamine (5-HT)-induced subcutaneous inflammation (Oliveira et al., 2007) or by PMN depletion preemptive to sciatic transection (Perkins and Tracey 2000), raising the possibility that PMN targeting of peripheral neurons can lead to hyperexcitability of ‘pain-sensing’ DRG neurons (nociceptors).

Recently, Nguyen et al. (2007) reported that a PMN-conditioned medium is sufficient to cause rapid (within 2 h) and significant loss of dissociated DRG neurons in culture, whereas Dinkel et al. (2004) demonstrated a delayed reduction in the viability of primary hippocampal neurons 3 days after co-culture with PMN in a cell contact dependant manner. However, the functional effects of PMN on neuronal activity and/or excitability remain unexplored. Therefore, this study was undertaken to characterize the activation state of PMN exposed to DRG cells, to determine the range of PMN titers that significantly reduce DRG neuronal count in culture, and to examine the effect of PMN on DRG neuronal excitability.

Our results indicate that co-incubation of PMN with DRG cells triggers activation of PMN which is detrimental to the viability of neurons, induces neuronal calcium transients, and increases the excitability of nociceptors.

Section snippets

Materials and methods

Neuronal and PMN cells were obtained from adult (200–250 g) male Sprague–Dawley rats that were used according to procedures approved by the Institutional Animal Care and Use Committee at Rhode Island Hospital.

Statistical analysis

Two-tailed analyses were performed using parametric tests at the alpha significance level of 0.05. Statistical significance was computed using one-way ANOVA or three-way ANOVA for analysis of variation in firing frequency with time at increasing current intensities. Tests of factors including pair-wise comparisons were performed with either unpaired or paired Student's t-test. In each cell loss experiment, PMN and DRG cells were obtained from 2 rats and co-incubated in at least 3 wells per

PMN activation

Within 5 min of addition of PMN (200 μl, 106 cells/ml) to a microwell coated with poly-D-lysine, laminin and collagen, prepared similarly as used for plating DRG cells and containing DMEM-F12 (2 ml), less than 5% of cells were fluorescently labeled with CM-DCF (data not shown), indicating a very weak intracellular ROS activity and therefore, predominantly resting PMN. However, more than 90% of cells manifested a clear fluorescent signal 2 min after addition of PMA, indicating their viability

Discussion

This study shows functional consequences of activated blood PMN on the viability, activity and excitability of DRG neurons. Using dissociated DRG neurons in culture, we demonstrate a significant decrease in neuronal count after co-incubation with activated PMN at a threshold E/T ratio between 0.2:1 and 0.4:1. Neuronal loss is probably unrelated to neuronal firing because lidocaine fails to abrogate this effect. The detrimental effect of PMN on neuronal viability does not necessarily require PMN

Acknowledgments

This study is supported by funds from Rhode Island Hospital and Rhode Island Foundation (CYS), and by NCRR/NIH P20 RR018728 and Rhode Island Foundation (SKS). The authors thank Derek M. Kozikowski for conceptualizing and writing the software used to analyze calcium transients and Zachary Jaffa for rodent handling.

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