Eur Spine J 2016 25(5): 1417-1427. Doi: 10.1007/s00586-015-4325-z
Plasmacytoid dendritic cells and memory T cells infiltrate true sequestrations stronger than subligamentous sequestrations: evidence from flow cytometric analysis of disc infiltrates
Andrea Geiss1, Rolf Sobottke2, Karl Stefan Delank3, Peer Eysel4
1 Department of Psychology, Biological Psychology, University of Cologne, Bernhard-Feilchenfeld-Straße 11, 50969 Cologne, Germany
2 Department of Orthopaedic Surgery, Medizinisches Zentrum StädtRegion Aachen, Mauerfeldchen 25, 52146 Würselen, Germany
3 Department of Orthopaedics and Traumatology, University of Haale/Saale, Magdeburgerstraße 22, 06112 Halle, Germany
4 Department of Orthopaedics and Traumatology, University of Cologne, Kerpener Straße 62, 50937 Cologne, Germany
Herniated nucleus pulposus has been considered to induce an adaptive immune response. Antigen recognition by antigen-presenting cells (APCs) represents an important step within manifestation of an adaptive immune response. Macrophages have been assumed to function as APC, while importance of plasmacytoid dendritic cells for initiation of an immune response directed towards herniated nucleus pulposus has never been examined. The aim of the present study was to assess importance of plasmacytoid dendritic cells for initiation of immune response directed towards herniated discs.
Fifteen patients with true sequestrations and three patients with subligamentous sequestrations underwent surgery after their neurological examinations. Disc material was harvested, weighted and digested for 90 min. Separated single cells were counted, stained for plasmacytoid dendritic cells (CD123+CD4+), macrophages (CD14+CD11c+) and memory T cells (CD4+CD45RO+) and analysed by flow cytometry. Both patient groups were compared in cell proportions. Furthermore, patients with true sequestrations (TRUE patients) were subdivided into subgroups based on severity of muscle weakness and results in straight leg raising (SLR) test. Subgroups were compared in cell proportions.
Plasmacytoid dendritic cells and memory T cells infiltrated true sequestrations stronger than the subligamentous sequestration and plasmacytoid dendritic cells predominated over macrophages in true sequestrations. Highest proportions of plasmacytoid dendritic cells were detected in infiltrates of patients having true sequestrations, severe muscle weakness and negative result in SLR test.
The findings of the present study indicate that plasmacytoid dendritic cells are involved in initiation of an immune response directed towards herniated nucleus pulposus, while macrophages may reinforce the manifested immune response and mediate disc resorption.
Keywords: Plasmacytoid dendritic cells; macrophages; motor weakness; sequestration; nucleus pulposus; memory T cells
Low back pain (LBP) has been identified as the third leading causes of years lived with disability . Low back-related leg pain or sciatic pain has been described as a form of LBP and is caused by herniated disc. Neurological deficits have been also reported as frequent symptoms of herniated disc.
Patients with disc-related sciatica are treated conservatively at first and thereafter surgically when conservative treatment fails. Efficiency of conservative treatment has been related to disc resorption . Although disc surgeries are conducted frequently, a significant proportion of patients complain of ongoing neurological deficits or sciatic pain after surgical treatment .
Herniated disc has been assumed to cause symptoms by three pathophysiologic mechanisms. These ones comprise mechanical compression of the spinal nerve roots by herniated disc tissue, production of hyperalgesic substances such as tumor necrosis factorα (TNFα) by nucleus pulposus and an autoimmune response directed towards herniated nucleus pulposus [4,5,6]. The autoimmune hypothesis is mainly derived from the unaccessibility of nucleus pulposus-derived antigens to lymphoid organs during the establishment of immunological tolerance, as this part of the intervertebral disc is not vascularized . Hence, nucleus pulposus has been suggested to be treated as foreign, if exposed to the immune system in association with disc herniation. The proteoglycan aggrecan has been suggested to represent the antigenic structure of nucleus pulposus .
An immune response directed towards herniated nucleus pulposus is initiated by antigen-presenting cells (APCs). An immune response is initiated by capturing and processing the antigen as well as stimulating naïve CD4+T cells to differentiate into effector CD4+T cells .
Macrophages and dendritic cells have been described as two types of APCs. Two subsets of dendritic cells have been characterized in humans, termed myeloid and plasmacytoid dendritic cells (PDCs). Although both APC types capture and process antigens, solely plasmacytoid dendritic cells are able to stimulate the differentiation of naïve CD4+T cells into effector CD4+T cells. Macrophages are incapable of triggering activation of naïve CD4+T cells .
Macrophages have been assumed to initiate an immune response directed against herniated nucleus pulposus. This assumption is mainly derived from reported immunopositivity of herniated disc tissue for surface marker CD68 [10,11]. Since surface marker CD68 is expressed by both macrophages and PDCs, observed immunopositivity of herniated disc tissue for CD68 may be also indicative of involvement of plasmacytoid dendritic cells in initiation of an immune response directed against herniated nucleus pulposus .
Involvement of plasmacytoid dendritic cells in initiation of an immune response directed against herniated nucleus pulposus implies stronger infiltration of disc tissue with PDCs as compared with macrophages. This assumption also implies stronger infiltration of disc tissue having provoked a stronger immune response with PDCs in comparison with disc tissue having induced a weaker immune response.
True and subligamentous sequestrations represent two subtypes of disc herniation. Disc tissues being classified as true sequestrations are thought to be exposed directly to the immune system. In contrast, disc tissues being classified as subligamentous sequestrations are considered to be not exposed to the immune system.
In more detail, true sequestrations refers to disc material which had penetrated the outer layers of the annulus fibrosus and the posterior longitudinal ligament, lying free in the epidural space. Disc tissues which lie free in the epidural space are thought to be exposed directly to the immune system, thereby provoking a strong immune response.
Subligamentous sequestrations refer to disc material which had passed through the annulus fibrosus, lying underneath the posterior longitudinal ligament . Posterior longitudinal ligament is thought to protect disc tissues from exposure to the immune system in the epidural space, thereby inducing a weak immune response.
To examine our hypothesis, we collected disc material from 15 patients with true sequestrations and three patients with subligamentous sequestrations immediately after their surgical treatment.
For tissue processing we developed a new technique enabling separation of a single cell-suspension from each resected disc material. Separated cells were stained for CD14+CD11c+macrophages, plasmacytoid CD123+CD4+dendritic cells and memory CD4+CD45RO+T cells and analyzed by flow cytometry.
We found that disc infiltrates of true sequestrations contained elevated proportions of plasmacytoid CD123+CD4+dendritic cells and memory CD4+CD45RO+T cells as compared with disc infiltrates of subligamentous sequestrations. Similar low numbers of CD14+CD11c+macro-phages were detected both in true sequestrations and subligamentous sequestrations.
Predominance of PDCs in true sequestrations implies their involvement in initiation of an immune response directed against herniated nucleus pulposus. Revealed few CD14+CD11c+macrophages are indicative of presence of M1 macrophages in disc infiltrates. In more detail, macrophages have been characterized to comprise two subsets, termed classical activated M1 and alternatively activated M2 macrophages . M1 and M2 macrophages produce different groups of matrix metalloproteinases (MMPs) and thus differ in their functions  (Figure 1).
M1 macrophages have been demonstrated to mediate degradation of extracellular matrix by releasing MMP-2 and MMP-9 and serve as phagocytes by producing reactive oxygen species (ROS), for instance [9,15]. There is also immunohistochemical evidence reporting involvement of macrophages and MMPs in mediation of disc resorption [16,17]. Hence, it is conceivable that M1 macrophages mediate disc resorption by degrading extracellular matrix and phagocytosing sequestrated disc material.
M2 macrophages are believed to be main regulators of fibrosis by producing MMP12, for instance [9,15,18]. Fibrosis has been defined as second stage of tissue repair process. In more detail, tissue repair process has been described to comprise two stages, termed a regenerative stage and fibrosis. While regenerative stage of tissue repair is caused by an initial tissue damage, fibrosis is due to an adaptive immune response following repeated tissue damage, for instance . A regenerative stage of tissue repair is characterized by a controlled matrix synthesis through the extracellular matrix and resolution of inflammatory response. Key feature of fibrosis is a disruption of the restitutive function of extracellular matrix and hence uncontrolled matrix synthesis [19,20,21]. Epidural fibrosis has been identified as risk factor of failed disc surgery .
Plasmacytoid CD123+CD4+dendritic cells may promote disc resorption by stimulating differentiation of macrophages into M1 macrophages. In more detail, PDC’s release of interferon stimulates differentiation of macrophages into M1 macrophages. Furthermore, plasmacytoid dendritic cells may promote onset of fibrosis by inducing differentiation of naïve CD4+T cells into TH2 effector cells. TH2 effector cells produce mainly the anti-inflammatory cytokine interleukin-4 (IL-4). This cytokine is required by macrophages to switch from a M1-like phenotype to M2-like phenotype .
The importance of this study:
Macrophages have been thought to initiate an immune response directed against herniated nucleus pulposus. The findings of the current study do not support this hypothesis because plasmacytoid dendritic cells and not macrophages were found to infiltrate predominately true sequestrations.
Hence, current study uncovers involvement of plasmacytoid dendritic cells in initiation of an immune response directed against herniated nucleus pulposus. Detection of few M1 macrophages in disc infiltrates implies involvement of macrophages in resorption of disc material at a later stage instead of initiating an immune response directed against herniated disc tissue. It is conceivable that macrophages are involved additionally in onset of fibrosis after switching from a M1-like phenotype to a M2-like phenotype and after regression of disc tissue. Plasmacytoid dendritic cells may promote involvement of macrophages in disc resorption and onset of fibrosis by releasing interferon and stimulating differentiation from naïve CD4+T cells into TH2 effector cells, respectively.
In sum, the findings of the current study are meaningful as they (a) challenge classical view of macrophages as an initiator of an immune response directed towards herniated nucleus pulposus (b) substantiate the involvement of macrophages in mediating disc resorption and onset of fibrosis (c) can be applied to the discovery of alternative treatments of low back pain and other disc problems. For instance, current study implies efficiency of treatments targeting immune cells for relief of disc problems.
Figure 1. Scheme of interactions between plasmacytoid CD123+CD4+dendritic cells and macrophages in mediation of disc resorption and onset of fibrosis in association with disc sequestration. Pictures of a plasmacytoid dendritic cell, a classically activated macrophage (M1) and an alternatively activated macrophage (M2) have been obtained from .
Abbreviations: Degradation of ECM: Degradation of extracellular matrix; OX40L: Tumour necrosis factor receptor ligand; ROS: reactive oxygen species; NO: Nitric oxide; TH2 cells: TH2 effector cells; IL-4: Interleukin-4; MMPs: matrix metalloproteinases
The work was supported by grants from the German Research Foundation (GE 1232/5-1) and the University Hospital of Cologne, Department of Orthopaedics and Traumatology, D-50937 Köln, Germany.
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Andrea Geiss, PhD
Lecturer for Biological Psychology, University of Cologne, Bernhard-Feilchenfeld-Straße 11, 50969 Cologne, Germany;
E-mail address: email@example.com