Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues

Acute low back pain is among the most common reasons for which patients seek medical care, with estimates of annual economic impact as high as $200 billion in the US (1). Degeneration of the intervertebral disc (IVD) is a common cause of such pain and disability, characterized by anatomic, morphologic, and biochemical changes including altered expression of both matrix metalloproteinases and proinflammatory cytokines (2). Elevated levels of molecular mediators of inflammation have been described in pathologic disc tissue, increasing with grade of degeneration (3,4). Such findings have been observed for both interleukin-1 (IL-1) and tumor necrosis factor α (TNFα), both of which have established roles in regulating nitric oxide (NO) and prostaglandin production, metalloproteinase expression, and apoptosis, all changes that may contribute to progressive pathology of the IVD. The inflammatory and immune activation profile exhibited by a herniated disc may be distinct from that observed in IVD degeneration and may differ further among different extents of herniation from protrusion through sequestration. Histologic evaluation of herniated disc tissue has revealed prominent infiltration of inflammatory cells, most markedly macrophages (5–10). Surgical specimens of herniated disc tissue also exhibit a greater presence of TNFα, IL-1β, and IL-6 compared with autopsy control tissue specimens (3,4), and explant cultures demonstrate a heightened release of IL-6, IL-8, prostaglandin E2 (PGE2), and NO from herniated disc tissue compared with control tissue (11). Recent studies have also revealed greater IL-12 and interferon-γ (IFNγ) expression in herniated disc fragments compared with bulging discs that remain contained within the disc space by an intact anulus fibrosus (12). Most of these cytokines are macrophage products, which is consistent with the greater presence of macrophages documented for herniated disc tissues (9,10). These cytokines can promote lymphocyte activation and differentiation while also recruiting additional macrophages and activating phagocytosis and secretion of proteolytic enzymes. Activated Th1 lymphocytes produce IFNγ that assists in further macrophage recruitment and activation, and the mechanism eliciting the heightened IFNγ expression in herniated disc fragments may represent a specific immune response against herniated nucleus pulposus tissue. The immune privilege of the nucleus pulposus occurs by both vascular isolation and a biochemical phenotype with the Fas ligand causing infiltrating T lymphocyte apoptosis. Support for this theory is found in regional lymph node accumulation of lymphocytes after exposure to autologous nucleus pulposus (13), lymphocyte accumulation in the IVD after anulus fibrosus injury (6), and immunoglobulin and complement membrane attack complex deposition in human herniated disc tissue (14–16). Recent studies have revealed the importance of Th17 lymphocytes in a range of inflammatory pathologies and autoimmune diseases, including rheumatoid arthritis, Crohn’s disease, and multiple sclerosis (17,18). IL-17 is a proinflammatory cytokine produced primarily by Th17 lymphocytes activated in response to IL-23 production. IL-17 can promote inflammation by different signaling cascades, some modulated by other lymphocyte products such as IFNγ (19), and thereby promote the synthesis of other cytokines, proteases, NO, and PGE2. Given the demonstrated presence of infiltrating and activated lymphocytes in herniated disc fragments and the potential importance of immune system activation by herniated disc tissues, it is of interest to determine a potential role of Th17 lymphocytes in mediating this process. The objective of this study was to evaluate differences in the immunophenotype of the IVD fragments obtained from patients undergoing surgery for treatment of degenerative disc disease and lumbar disc herniation. The presence of Th17 lymphocyte markers in IVD pathology has not yet been investigated, and a primary goal of this study was to use immunohistochemistry to evaluate the molecular markers of different inflammatory and immune cells alongside expression of the lymphocyte product IL-17 in this surgically obtained tissue. Macrophage and lymphocyte markers as well as other mediators of inflammation known to be of relevance to IVD disease, including IL-6, IFNγ, IL-12, IL-10, and IL-4, were also evaluated to more comprehensively characterize the inflammation pattern. All markers were evaluated in degenerated and herniated disc fragments as well as in cadaveric nucleus pulposus and anulus fibrosus tissues with little to no evidence of degeneration. Results of this study reveal a high expression level of IL-17 in surgical tissues obtained from both degenerated and herniated IVDs, but not from autopsy tissue, suggesting a role of Th17-mediated inflammatory processes in IVD pathology. Additional results reveal the heightened presence of immune-activating IFNγ in herniated disc fragments, with concomitant greater macrophage infiltration and IL-6 expression as compared with degenerated tissues. These findings illustrate a pattern of immune activation by herniated disc tissue involving macrophage infiltration and activation, and further suggest that a novel lineage of Th17 lymphocytes may also be involved in both forms of IVD pathology.