Abstract Study Design. Cultures established from murine disc derived cells were stimulated by Lipopolysaccharide (LPS). Cells capacity to secrete pro-inflammatory cytokines and IL-10 with and without LPS stimulation was determined using ELISA assays. Objectives. To determine the capacity of disc derived cells to secrete pro-inflammatory cytokines, and the effect of LPS stimulation on the secretion. Summary of Background Data. The pathophysiology of compressive radiculopathy is unclear. Inflammation is contemplated as a possible explanation. Pro-inflammatory cytokines secretion was demonstrated in herniated nucleus pulposus (HNP). It is unknown whether these cytokines are secreted from disc derived cells or from infiltrating inflammatory cells in HNP. Methods. Discs were microsurgically harvested from inbred mice and cut, to allow nucleus pulposus (NP) to extrude and establish cell culture. A study group was exposed to LPS stimulation. Media were harvested from the study and control groups 24 hours later. Secretion of IL-1-$, IL-6, IL-10, GM-CSF and TNF-a were determined using ELISA assays. Results. Basal secretion of IL-6 and IL-10, but no basal secretion of IL-1-ß, GM-CSF or TNF-a were detected. Secretion of IL-1-$ and GM-CSF was detected following LPS stimulation, as well as a 75-fold increase in IL-6 secretion and a 150-fold increase in IL-10 secretion. No TNF-a secretion was detectable. All result had high statistical significance. Conclusions. Cultured murine disc derived cells have the capacity to secrete pro-inflammatory cytokines and IL-10 in the absence of inflammatory cells. This finding supports the hypothesis that disc derived cells are capable of initiating or amplifying an inflammatory process. [Key words: cytokines, intervertebral disc, radiculopathy, inflammation, disc herniation]

Mini Abstract Pro-inflammatory cytokines are secreted by the inflammatory tissue around herniated nucleus pulposus, but it is unclear whether this secretion is from disc derived cells or from the inflammatory infiltrate. Murine disc derived cell cultures were used to determine the capacity of these cells to secrete pro-inflammatory cytokines, with and without lipopolisaccharide stimulation. Murine disc derived cell cultures were found capable of secreting IL-1-beta, IL-6, IL-10 and GM-CSF, but not TNF-a, in the absence of inflammatory infiltrate. This secretion increased following lipopolysaccharide stimulation. These findings support a role for disc derived cells in the pathophysiology of inflammation in HNP.

Introduction The pathogenesis of radiculopathy was traditionally attributed to nerve root compression by a fragment of a herniated disc, as described by Mixter and Barr24. It is well known however, that a nerve root mechanically compressed by herniated nucleus pulposus (HNP) becomes at times edematous, and inflamed 3,12,33. It was therefore contemplated that inflammation around HNP may be an important factor in the pathophysiology of radiculopathy2,5,8,21,22,25,30,35,38. This hypothesis is supported by histological, biochemical, and functional data collected in experiments which involved exposure of the cauda equina and the epidural space to nucleus pulposus (NP). These studies demonstrated the presence of inflammatory infiltrate in and around HNP tissue, as well as the dorsal nerve root following exposure to NP or to cells derived from NP. The studies showed also secretion of inflammatory mediators and changes in the electrical conductance through the affected nerve root3,5,17,23,27,38. Several different mechanisms were offered as possible explanation for inflammation in HNP. Biochemical compounds which are irritating to the nerve root and are present in the NP (such as Phospholipase A2) were considered5,30. Others suggested an auto-immune mechanism, where the immune system fails to recognize NP derived antigens as “self” when extruded from the isolated, a-vascular disc to a vascular environment2,7. Systemic auto-immune phenomena were not demonstrated however in patients with HNP. It is quite clear that the inflammation in HNP has an immunological component as demonstrated by the presence of immune competent cells in the reaction around HNP8-10,28,30. Regardless of the precise inflammatory mechanism, inflammatory mediators and regulators were found to be present in large quantities in HNP. These include, among others, IL-6, NO, and neutral metalloproteases14-16. The intact NP is a-vascular. Upon disc injury and exposure of its contents to an extra-discal, vascular environment, neo-vascularization occurs into the extruded disc material, allowing inflammatory cells to migrate into the HNP through the endothelium of the neo-vascularization 36,37,39. It is therefore unclear whether the cytokines secretion measured in HNP is derived from inflammatory cells which infiltrated the HNP tissue, or from disc derived cells. Such knowledge may shed more light on the mechanism by which inflammatory response is initiated in HNP, and on the pathophysiology of compressive radiculopathy. The purpose of this study was to determine the capacity of disc derived cells to secrete pro-inflammatory cytokines (IL-1-ß, IL-6, TNF-a, GM-CSF) and IL-10 - a cytokine involved in the regulation of pro-inflammatory cytokines production.

Methods Murine spines were harvested from C57Bl6 male mice aged 6 - 8 weeks, immediately following euthanasia. Discs were microsurgically removed and kept in Dulbecco’s modified Eagle’s medium (DMEM) (Beit-Haemek, Israel). Discs were collected from all available lumbar and thoracic levels (cervical discs were too small to handle). The discs were sharply cut and laid on plastic culture dishes (Nunc) containing DMEM, supplemented by 10% fetal calf serum (FCS) (Beit-Haemek, Israel). All disc cultures were established within two hours of harvesting. Cultures were kept in a humidified incubator, saturated by 5% CO2 at 370 C. All protocols were carried out in compliance with the guidelines of the animal care committee of the Hebrew University- Hadassah Medical School, and under its approval. On light microscopy, cultures comprised mainly of small round nucleated cells - probably of chondrocytic lineage, which developed 2-6 membrane processes during adherence, and were actively dividing. Another cell sub-population of large, round multinucleated cells was also noted - probably notochord cells. Ten days later, when cell adherence was achieved, the medium was replaced with a fresh, serum free DMEM (to avoid undesired exposure of the cultures to stimulants possibly present in the FCS). 24 hours later, Lipopolysaccharide (LPS) from E.Coli (10µg/ml) (Sigma) was added to the study group dishes. Media were harvested 24 hours later and frozen at -80°C until determination of their cytokines contents. Cells were detached from the culture dish by a 15 minutes exposure to 0.25% trypsine (sigma), and counted, to enable later correction of the measured quantities of cytokines to the number of cells in each dish. Pro-inflammatory cytokines and IL-10 levels in the media were determined using ELISA assays (Genzyme). Each set of tests comprised of 50-60 discs harvested from 5 mice. Each dish contained 25-30 cut discs. This procedure was repeated 3 - 8 times, (some cytokines were not studied initially and therefore had less repetitions). Measured values were corrected for 105 cells, mean values were calculated, and Wilcoxon - Rank sum test was used to determine the statistical significance of the differences measured between the mean cytokines secretion in the control group and the study group.

Results Basal secretion of TNF-α, IL-1-ß and GM-CSF was undetectable in both the study and the control groups. A low mean basal secretion of IL-10 was detected (1.9 pg/105 cells), as well as a mean IL-6 basal secretion of 400 pg/105 cells. Stimulation with LPS caused secretion of IL-1-ß and GM-CSF, a 75-fold increase in IL-6 mean secretion, and a 150-fold increase in the mean secretion of IL-10. TNF-α was consistently undetectable in all cultures, regardless of LPS stimulation (table 1, Figures 1-4).

Discussion HNP contains inflammatory infiltrate from the surrounding vascularized environment. It is unclear whether the previously reported secretion of inflammatory mediators originates in NP derived cells or in inflammatory cells present in the neo-vascularized tissue which penetrates HNP. The purpose of this study was to determine whether murine disc derived cells are capable of secreting pro-inflammatory cytokines (IL-1-ß, IL-6, TNF-a, GM-CSF) and IL-10, and to study their response to stimulation with LPS. The results allow to conclude that cultured cells derived from intact murine disc have trace or no basal secretion of IL-1-ß and GM-CSF but are capable of secreting significant quantities of these cytokines following stimulation with LPS. Murine disc derived cultured cells have a basal secretion of IL-6 and IL-10 which increases significantly following LPS stimulation. The absence of TNF-α secretion may be explained by lack of TNF-α production capacity, or that another stimulus is required to induce its secretion. The model employed in the present study, was designed to exclude the possible effects of non disc derived cells on cytokines secretion. By studying intact discs, it can be safely assumed that the contribution of leukocytes, macrophages and other inflammatory cells to cytokines secretion is largely excluded. Although it is impossible to completely exclude a minimal contribution to cytokines secretion by a possible contamination of neighboring cells (such as osteoclasts or fibroblasts) during preparation, the study design allows to conclude that at least part of the cytokines secretion in HNP originates in disc derived cells and not only in inflammatory infiltrate. These findings have relevance to elucidating the pathophysiology of radiculopathy. Disc tissue was shown to contain proteolytic enzymes such as stromelysin, neutral metalloproteases and collagenase6,19,23,26,31. These enzymes were shown to be activated by pro-inflammatory cytokines in articular cartilage4,11,13,18,32. It was also shown that articular cartilage is capable of secreting pro-inflammatory cytokines, as well as expressing antigens which play a role in the induction and amplification of inflammation1,20,34. In the present study, disc derived cells were shown to secrete pro-inflammatory cytokines. It may therefore be possible that like in the articular cartilage, pro-inflammatory cytokine secretion is regulating proteolytic enzymes which may inflict local and neural damage, increase inflammation and cause clinical signs of radiculopathy. Elucidating the precise pathophysiology of radiculopathy requires further studies of the inflammatory mechanism in disc herniation, to better understand such possible connections, and to validate the findings in humans.

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Table 1: Pro-Inflammatory Cytokines Secretion by Nucleus Pulposus Cells in Culture, with and without LPS (pg/105 cells) Cytokine Mean Secretion (no LPS)LPS Free Culture Mean Secretion (with LPS)LPS stimulated Culture P value IL-1-$ 0 27.69 .0006 IL-6 399.7 29692.50 <.0001 IL-10 1.9 297.43 .001 GM-CSF 0 9.77 .001 TNF-" 0 0