Abstract
Aims
CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration.
Methods
We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry.
Results
We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues.
Conclusion
Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state.
Cite this article: Bone Joint Res 2023;12(3):189–198.
Article focus
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This study aimed to verify the pathophysiological relevance and mechanism of action of monomeric CRP (mCRP) in healthy and degenerative intervertebral disc (IVD) cells.
Key messages
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mCRP can be present in IVD tissues and induces the expression of multiple proinflammatory and catabolic factors in human annulus fibrosus (AF) and nucleus pulposus (NP) cells.
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The effect of mCRP is persistent and sustained, regardless of the proinflammatory environment, as it was similar in healthy and degenerative human primary AF cells.
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mCRP effects in healthy and degenerative AF cells are mediated by phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2), and nuclear factor-κβ (NF-κβ) p65 signalling pathways.
Strengths and limitations
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This is the first experimental article that provides evidence for the functional activity of mCRP in healthy and degenerative human AF and NP disc cells.
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This is the first article demonstrating the localization of mCRP in intravertebral disc cells of the AF and NP.
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Further studies are needed to confirm the role of mCRP in IVD degeneration. A larger number of subjects studied would give more strength to our results; the limited access to primary human disc cells is a limitation of this study.
Introduction
Low back pain (LBP) is the main cause of disability worldwide, affecting around 700 million people.1 It has been estimated that 70% to 85% of the global population has LBP at some time in their lives, and it can limit the activity of people under 45 years of age, generating a huge socioeconomic impact.2 The aetiology of LBP is complex, multifactorial, and still unclear. However, histological and MRI data associate chronic LBP with lumbar intervertebral disc (IVD) degeneration.3 IVDs are embedded between the vertebrae and provide flexibility to the spine. They consist of three anatomical parts: an inner cell-sparse gelatinous nucleus pulposus (NP), an outer fibrous region, the annulus fibrosus (AF), and, limiting above and below the AF, the hyaline cartilaginous end plates (CEPs).4 In a healthy adult IVD, homeostasis between extracellular matrix (ECM) synthesis and degradation is balanced by growth factors and cytokines. An imbalance towards catabolic processes contributes to structural degeneration of the IVD, likely contributing to the development of LBP.3,5
IVD degeneration leads to multiple anatomical, mechanical, and biochemical disc changes. These alterations decrease mechanical stability and shock absorber functions, contributing to osteophyte formation, annular fissures, and decreased motion of spinal segments.3 Genetic risk has an important influence on IVD degeneration, however mechanical trauma, injuries, smoking, obesity, and ageing also play a role.6 In response to inflammation and degeneration, vascular and nerve ingrowth into the avascular IVD occur from the outer layers of the AF, extending into the NP.7,8
IVD degeneration and the degeneration of joint cartilage as seen in osteoarthritis (OA) show marked similarities, although they are barely discussed simultaneously. Striking similarities are seen on plain radiographs: the loss of disc height or joint space, sclerosis of the subchondral bone, and the development of osteophytes. Furthermore, in both IVD and joint cartilage, the ECM is composed of similar constituents (although in another ratio), and similar ECM-degrading machinery is present in the process of degeneration. There are also differences such as the type of forces applied to the ECM, and the absence of synovial fluid in IVDs.9-12
CRP is an acute-phase inflammatory protein whose plasma levels sharply increase in response to injury, infection, and inflammation. It is a member of the pentraxin family, and circulates prevalently in blood in its homopentameric form.13 CRP plasma levels increase from around 1 µg/ml to over 500 µg/ml within 24 to 72 hours upon severe tissue damage.14 Thus CRP levels are a well-established biomarker for monitoring the inflammatory status in infections and other multiple diseases, including IVD herniation15 and rheumatic diseases such as OA.16
The pentameric protein is characterized by a discoid configuration of five identical non-covalently bound subunits of about 23 kDa each. It is synthesized primarily in liver hepatocytes but has also been reported to be produced in other cell types such as smooth muscle cells,17 macrophages,18 endothelial cells,19 lymphocytes, and adipocytes.20 The monomeric CRP (mCRP) differs from the pentameric CRP (pCRP) in its different antigenic, biological, and electrophoretic mobility,21 and in its expression of different neoepitopes.22 These two distinct forms of CRP showed distinct biological functions in the inflammatory process. It has been proved that pCRP suppresses the adherence of platelets to neutrophils, whereas mCRP enhances these interactions.22 This difference in function can be explained by the two isoforms binding to differing types of Fcγ-receptors involved in the signalling process. The mCRP isoform uses the low-affinity immune complex binding immunoglobulin G (IgG) receptor called FcγRIIIb (CD16b) on neutrophils and FcγRIIIa (CD16a) on monocytes, while pCRP binds to the low-affinity IgG receptor FcγRIIa (CD32).23 Evidence shows that pCRP tends to exhibit weak anti-inflammatory activities. In contrast, mCRP has marked proinflammatory properties both in vitro and in vivo by promoting monocyte chemotaxis and the recruitment of circulating leucocytes to areas of inflammation via Fcγ-RI and Fcγ-RIIa signalling.24,25 However, the pathophysiological role of CRP as a regulator of inflammatory processes remains obscure.
Some of the published IVD disease literature assesses CRP as a reference biomarker of an ongoing inflammatory process.15,26,27 Ruiz-Fernández et al24 recently investigated the inflammatory effect of mCRP in chondrocytes, proving that mCRP triggers a sustained proinflammatory and catabolic state in healthy and OA cartilage. However, the role of mCRP in disc pathophysiology has not yet been clarified. In this current study, we evaluated the functional role of mCRP as a modulator of the inflammatory response in NP and AF human cells, assessing some proinflammatory and catabolic factors and the pathways involved in the signal transduction upon mCRP stimulation.
Methods
Reagents
Fetal bovine serum (FBS), lipopolysaccharide (LPS), and human recombinant interleukin (IL)-1β were obtained from MilliporeSigma (USA). Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 medium, trypsin-ethylenediaminetetraacetic acid, L-glutamine, and penicillin-streptomycin mixture were obtained from Lonza Group (Switzerland). Pronase and collagenase P were obtained from Roche Diagnostics (Germany).
Recombinant mCRP (Roosevelt University, College of Pharmacy, USA) was produced in Escherichia coli, had both cysteines replaced with alanine residues and solubilized by an acylation technique (C-rmCRP) in 25 mM NaPBS (pH7.4), and was purified and tested in several bioassays to confirm its equivalence to biological mCRP from the pentameric form and free of contaminating endotoxin. The final endotoxin level of all protein solutions was below the detection limit 0.06 endotoxin units (EU)/ml of the assay.28
Cell culture conditions and treatments
Human NP immortalized cell line HNPSV-129 (Tokai University School of Medicine; Japan) was cultured in DMEM/Ham’s F12 supplemented with 10% FBS, 4 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. In these experiments we used a cell passage range of 13 to 22.
Healthy IVD samples were obtained from deceased multi-organ donors. Degenerative IVD samples were obtained, under written patient consent, from patients undergoing discectomy. Inclusion criteria were: male or female patients undergoing discectomy diagnosed with spinal stenosis; adult degenerative scoliosis; disc herniation; and disc degeneration (Pfirrmann grade > 3 to 4). Exclusion criteria were: previous spinal malignancies or infections; previous spinal surgery; and patients diagnosed with metabolic diseases. All IVD samples were obtained under permission from the local ethics committee, according to the Declaration of Helsinki.30 AF cells were isolated and cultured as previously described.31 Briefly, aseptically dissected AF tissue was diced and properly rinsed with phosphate-buffered saline (PBS). After removing PBS, tissue was incubated for 20 minutes with pronase at a final concentration of 1 mg/ml in DMEM/Ham’s F12 at 37°C. Then, pronase was removed and the tissue was rinsed two to three times with PBS. After removing PBS, digestion continued with collagenase P at a final concentration of 1 mg/ml in DMEM/Ham’s F12 with 10% FBS. Tissue was incubated for four to six hours in agitation at 37°C. The resulting cell suspension was filtered with a 40 μm nylon cell strainer (BD Biosciences Europe, Belgium) to remove debris. Cells were centrifuged, washed with PBS, resuspended in DMEM/Ham’s F12 supplemented with 10% FBS, 4 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin, counted, and plated in 12-well culture plates. Cells were maintained in culture for a maximum of ten days for expansion. All AF cells used in these experiments were at passage number 2.
AF cells and HNPSV-1 cells were seeded in six-well plates (2.5 × 105 cells/well) and treated with 10, 25, or 50 µg/ml mCRP, 100 ng/ml LPS, or 0.1 ng/ml IL-1β, after serum starvation for four hours. All the concentrations were selected based on a previous study24 and in the absence of IVD cell toxicity. All treatments were done in serum-free conditions after four hours of serum starvation and lasted 24 hours for the proinflammatory response experiments (Figures 1 to 3), or 15 minutes for the intracellular signal transduction experiments (Figure 4).
Fig. 1
Monomeric CRP (mCRP) proinflammatory effect on human primary degenerative intervertebral disc (IVD) annulus fibrosus (AF) cells. Human primary AF cells derived from degenerative IVD in culture were challenged with 10 or 50 µg/ml mCRP, or with 100 ng/ml lipopolysaccharide (LPS) as positive control for 24 hours, after four hours of serum starvation. a) to g) Left panels. Messenger RNA (mRNA) expression was determined by quantitative real-time polymerase chain reaction. mRNA levels are presented as fold change relative to control (C-). a) to g) Middle and right panels. Protein expression was measured by western blot. Data from densitometric analyses normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are shown as relative-to-control (C-) values. Data are expressed as mean (standard error of the mean) of at least three independent experiments. Comparisons referred to control (C-): *p < 0.05, **p < 0.01, ***p < 0.001. Cal., calibrator (refers to control (C-) condition); COX2, cyclooxygenase 2; IL, interleukin; LCN2, lipocalin 2; MMP13, matrix metalloproteinase 13; NOS2, nitric oxide synthase 2; PTGS2, prostaglandin-endoperoxide synthase 2; VCAM1, vascular cell adhesion molecule 1.
Fig. 2
Monomeric CRP (mCRP) proinflammatory effect on human primary healthy intervertebral disc (IVD) annulus fibrosus (AF) cells. Human primary AF cells from healthy IVD in culture were challenged with 10 or 50 µg/ml mCRP, or with 100 ng/ml lipopolysaccharide (LPS) as positive control for 24 hours, after four hours of serum starvation. a) to g) Messenger RNA (mRNA) expression was determined by quantitative real-time polymerase chain reaction. mRNA levels are presented as fold change relative to control (C-). Data expressed as mean (standard error of the mean) of at least three independent experiments. Comparisons referred to control (C-): *p < 0.05, **p < 0.01, ***p < 0.001. Cal., calibrator (refers to control (C-) condition); IL, interleukin; LCN2, lipocalin 2; MMP13, matrix metalloproteinase 13; NOS2, nitric oxide synthase 2; PTGS2, prostaglandin-endoperoxide synthase 2; VCAM1, vascular cell adhesion molecule 1.
Fig. 3
Monomeric CRP (mCRP) proinflammatory effect on human intervertebral disc (IVD) nucleus pulposus (NP) cell line. HNPSV-1 human NP cells were challenged with 10, 25, or 50 µg/ml mCRP, or with 100 ng/ml lipopolysaccharide (LPS) or 0.1 ng/ml IL-1b as positive controls for 24 hours, after four hours of serum starvation.a) to f) Messenger RNA (mRNA) expression was determined by quantitative real-time polymerase chain reaction. mRNA levels are presented as fold change relative to control (C-). Data expressed as mean (standard error of the mean) of at least three independent experiments. Comparisons referred to control (C-): *p < 0.05, **p < 0.01, ***p < 0.001. Cal., calibrator (refers to control (C-) condition; IL, interleukin; LCN2, lipocalin 2; MMP13, matrix metalloproteinase 13; PTGS2, prostaglandin-endoperoxide synthase 2; VCAM1, vascular cell adhesion molecule 1.
Fig. 4
Mediators involved in monomeric CRP (mCRP) intracellular signal transduction in human primary healthy and degenerative intervertebral disc (IVD) annulus fibrosus (AF) cells. Human primary AF cells from healthy or degenerative IVD in culture were challenged with 50 µg/ml mCRP or with 100 ng/ml lipopolysaccharide (LPS) as positive control for 15 minutes, after four hours of serum starvation. a) to f) Protein expression was measured by western blot. Determination of the phosphorylated protein normalized to total protein and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from densitometric analyses are shown as relative-to-control (C-) values. Data expressed as mean (standard error of the mean) of at least two or three independent experiments. Comparisons referred to control (C-): *p < 0.05, **p < 0.01, ***p < 0.001. NF-κβ p65, nuclear factor-κβ p65; pERK1/2, phosphorylated extracellular signal-regulated kinase 1/2; PI3K, phosphoinositide 3-kinase; pPI3K, phosphorylated PI3-kinase; pNF-κβ p65, phosphorylated nuclear factor-κβ p65.
Immunohistochemical assays
Sections 4 μm thick obtained from human AF or NP tissue were mounted on silanized coated slides (Dako-Agilent; USA). Epitope retrieval was performed in a PT-Link (Dako-Agilent) at high pH for 20 minutes. Then, the slides were automatically immunostained in an Autostainer-Link 48 (Dako-Agilent), employing the monoclonal anti-CRP antibody, clone CRP-8 (MilliporeSigma), diluted 1:100, and 30-minute incubation time. As a detection system, we used EnVision FLEX/HRP (Dako-Agilent), for 30 minutes, and 3,3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen, for five minutes.
RNA isolation and real-time reverse transcription-polymerase chain reaction
Total RNA was isolated from cell culture with NZYol (NZYTech, Portugal) and E.Z.N.A. Total RNA Kit I (Omega Bio-tek, USA) according to the manufacturer’s instructions, and reverse-transcribed using NZY First-Strand cDNA Synthesis Kit (NZYTech). Then, SYBR-green-based quantitative real-time polymerase chain reaction (RT-qPCR) was performed in Stratagene MX3005P thermal cycler as previously described,32 using a standard protocol with RT2 SYBR Green qPCR Mastermix and specific PCR primers (Qiagen, Germany) (human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 175 bp, PPH00150E, reference position 1287, Gen-Bank accession no. NM_002046.3; human nitric oxide synthase 2 (NOS2), 132 bp, PPH00173E, reference position 3962, Gen-Bank accession no. NM_000625.4; human matrix metalloproteinase 13 (MMP13), 61 bp, PPH00121B, reference position 1380, Gen-Bank accession no. NM_002427.3; human vascular cell adhesion molecule 1 (VCAM1), 141 bp, PPH00623E, reference position 2980, Gen-Bank accession no. NM_001078; human Lipocalin 2 (LCN2), 87 bp, PPH00446E, reference position 626 to 644, Gen-Bank accession no. NM_005564.3; human IL-6, 98 bp, PPH00560C, reference position 816, Gen-Bank accession no. NM_000600.3; human IL-8, 126 bp, PPH00568A, reference position 326, Gen-Bank accession no. NM_000584.3; human prostaglandin-endoperoxide synthase 2 (PTGS2), 63 bp, PPH01136F, reference position 1502, Gen-Bank accession no. NM_000963.2). No-template controls were included to eliminate any non-specific amplification, and melting curves were generated to ensure a single gene-specific peak. Gene expression changes were determined by the comparative ΔΔCt method in MxPro qPCR Software version 4.10 (Stratagene, USA), and expressed as relative fold changes compared to control (C-) and normalized to GAPDH housekeeping gene.
Protein extraction and western blot analysis
After treatment, cells were rapidly washed with ice-cold PBS and scraped in lysis buffer (10 mM Tris–HCl, pH 7.5, 5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF)), freshly supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, USA), then the total cell lysates were centrifuged at 14,000 g for 20 minutes. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotting procedures were performed as previously described.33 Immunoblots were incubated with the specific antibody against NOS2, cyclooxygenase 2 (COX2), phospho-phosphoinositide 3-kinase (PI3K) p85/p55, phospho-p44/p42 MAPK (extracellular signal-regulated kinase 1/2 (ERK1/2)), phospho-nuclear factor-κβ (NF-κβ) p65, or NF-κβ p65 diluted 1:1000 (Cell Signaling Technology, USA), PI3K p85 or MAPK 1/2 (ERK1/2) diluted 1:1000 (MilliporeSigma), VCAM1 diluted 1:500 (EnoGene Biotech, USA), IL-6 or IL-8 diluted 1:100 (Santa Cruz Biotechnology, USA), MMP13 diluted 1:500 (Abcam, UK), and human LCN2 diluted 1:1,000 (R&D Systems, USA).
The immune complexes were detected using anti-rabbit, anti-mouse (both from GE Healthcare, USA), or anti-goat (Santa Cruz Biotechnology) horseradish-peroxidase-labelled secondary antibodies diluted 1:5,000 and visualized with Immobilon Western Detection kit (Millipore, USA). To verify equivalent protein loading, the membranes were stripped 15 minutes in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7), blocked and incubated with anti-GAPDH antibody diluted 1:2,000 (MilliporeSigma). In all figures showing images of gels, the bands for each picture were obtained from the same gel, although they may have been spliced for clarity. The images were captured with ChemiDoc MP Imaging System and analyzed with Image Lab 6.0.1 Software, both from Bio-Rad Laboratories (USA)
Statistical analysis
Data are reported as mean (standard error of the mean (SEM)) of at least three independent experiments. Statistical analyses were performed with GraphPad Prism 9.3.1 software (GraphPad Software, USA). Assuming a normal distribution, one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test were employed. As we performed few planned comparisons, p-values have not been corrected for multiple comparisons; they apply individually to each value reported and not to the entire family of comparisons. A p-value less than 0.05 was considered to be statistically significant.
Results
As shown in Figure 1, mCRP increases IL-6, IL-8, LCN2, VCAM1, NOS2, COX2, and MMP13 mRNA expression and protein levels in cultured human primary AF cells from patients with IVD degeneration, as confirmed by RT-qPCR and western blot analysis. The induction of proinflammatory mediators occured in a dose-dependent manner after 24 hours.
The upregulation of these inflammatory and catabolic factors by mCRP was also determined in human primary AF cells from healthy patients, suggesting that mCRP can modify the healthy cell status by promoting the development of a proinflammatory environment in the IVD (Figures 2a to 2g). In all the experiments with human cells, LPS inflammatory stimulus was used as control of inflammatory induction.
To broaden the scope of our results, we also tested mCRP activity in HNPSV-1, a cell line derived from human NP cells that maintains the original architecture of the cells and their gene expression profile.29Figure 3 shows that mCRP at 10 μg/ml, 25 μg/ml, and 50 μg/ml significantly increases IL-6, IL-8, LCN2, VCAM1, PTGS2, and MMP13 mRNA expression levels, as previously observed in human primary AF cells. LPS and IL-1β, as well-known inflammatory stimuli, were used as controls of inflammatory induction.
For completeness, to elucidate which mediators might be implicated in mCRP intracellular signal transduction in both healthy and degenerative human disc cells, we analyzed NF-κβ signalling pathway upon mCRP challenge. Healthy or degenerative human primary AF cells were treated with 50 μg/ml mCRP or 100 ng/ml LPS as a positive control, for 15 minutes. The results obtained showed that mCRP induces PI3K phosphorylation, ERK1/2 phosphorylation, and NF-κβ p65 phosphorylation in healthy AF cells (Figures 4a to 4c) and degenerative AF cells (Figures 4d to 4f).
Finally, we performed immunohistochemical assays to investigate whether mCRP is located in IVD tissues. We assessed mCRP presence in human degenerative and healthy AF and healthy NP tissues, confirming its presence in physiological state in all of them. As we can observe in Figures 5a to 5c, mCRP location is predominantly cytoplasmic and perinuclear, being the more intense stain in the AF, especially in the degenerative tissue, than in the NP tissue.
Fig. 5
Monomeric CRP (mCRP) localization in human degenerative and healthy intervertebral disc tissues. Immunohistochemical images showing mCRP protein presence (brown staining) in: a) human degenerative annulus fibrosus (AF) tissue; b) human healthy AF tissue; and c) human healthy nucleus pulposus (NP) tissue. Images taken at 60× magnification.
Discussion
CRP in its pentameric form is an acute-phase serum protein and a well-established biomarker in a great number of infectious and inflammatory diseases, including IVD herniation.34 It has also been evidenced as a mediator of inflammatory and immune responses in the context of local tissue injury.35,36 Serial CRP measures are useful not only for the follow-up of postoperative infection, but also for monitoring treatment efficacy and for the detection of relapses.27,37
CRP monomeric isoform, although much less studied, has been found to have different biological functions in the inflammatory process. While clarifying its pathophysiological function needs further investigation, new insights could be extremely helpful in better understanding the inflammatory mechanism and how it works in the pathogenesis and disease progression.
There is evidence that the inflammatory process plays an important role in the development of disc degeneration, and there is a marked correlation between the levels of inflammatory markers and the degree of degeneration.38 An association has been previously reported between high-sensitivity CRP (hs-CRP) levels and severe pain (VAS of > 40).39 Sugimori et al40 found markedly higher mean hs-CRP levels among patients with lumbar disc herniation (0.056 mg/dl (standard deviation (SD) 0.076)) than in the control group (0.017 mg/dl (SD 0.021)); no significant correlation between hs-CRP levels and herniation level was reported, but those patients with a higher concentration of hs-CRP before operation showed a poorer recovery. Of interest, some papers only quantified CRP levels in the high sensitivity range (i.e. hs-CRP), describing values below 10 μg/ml. According to several studies41-47 and to the guidance of the USA Food and Drug Administration (FDA),48 the clinical significance of such values is questionable.
In cervical disc herniation, pain occurs as a result of the release of local inflammatory cytokines. The elevated plasma concentration of IL-6 produced by macrophages around the IVD tissue increments CRP levels.49,50 It has been shown that CRP levels were considerably higher in patients with cervical disc hernia and neck pain compared with those patients with neck pain but a normal cervical magnetic resonance.34 A recent study found that plasma CRP levels in peripheral blood did not differ between healthy controls and non-herniated IVD degeneration patients.51
Recently, it has been demonstrated that only mCRP originated from local pCRP in the joint compartment may bind to receptor activator of nuclear factor-κB ligand (RANKL), neutralizing its activity and downregulating osteoclast differentiation.52 Ruiz-Fernández et al24 recently reported the inflammatory effect of mCRP in chondrocytes, demonstrating that mCRP triggers a sustained proinflammatory and catabolic state in OA and healthy cartilage, and that the NF-κβ pathway is involved in its signal transduction. To demonstrate that this activity can also be exerted in IVD cells, we tested the effect of mCRP in primary culture of human AF cells isolated from patients with IVD degeneration or from healthy subjects. In order to show that these effects are not exclusive to AF cells, the human NP immortalized cell line HNPSV-1 was used as a NP cells model for comparison in order to study mCRP effects in the whole disc, since NP cells commonly exhibit chondrocyte-like characteristics.53
As far as we know, this is the first experimental article that provides evidence for the functional activity of mCRP in AF and NP disc cells. To gain further insights into the action of mCRP and to verify whether this stimulation can result in sustained proinflammatory effects, we evaluated the dose-dependent activity of mCRP in master inflammatory and catabolic players in IVD degeneration. IL-8 and IL-6 are two of the proinflammatory cytokines strongly expressed in IVD degeneration.54 In this work, we explored the ability of mCRP to modulate some of the most important factors involved in the disc degeneration inflammatory and catabolic processes. We also assessed LCN2, an adipokine implicated in the catabolic mechanism of IVD degeneration, MMP13, a catabolic enzyme, and the inflammatory mediators NOS2, cyclooxygenase 2 (COX2), and VCAM1. Nitric oxide synthase type II (iNOS) and COX2 are classically involved in the amplification of inflammatory responses. NO has cytotoxic effects, damages the IVD, and is involved in the upregulation of MMPs, among which MMP13 is one of the major mediators of ECM degradation. COX2 induces prostaglandins that in turn increase the synthesis of other inflammatory mediators, including cytokines, to perpetuate tissue destruction. All these mediators are produced by IVD cells and are elevated in IVD degeneration.55-59
Our present findings bring out important evidence regarding the modulation of IVD metabolism and functions exerted by mCRP, as well as the localization of mCRP in human IVD tissues. First, exposure of AF and NP cells to mCRP induced multiple proinflammatory genes, suggesting that its effects are not limited to one specific molecular target, rather, mCRP has a broader spectrum of action. The second important point is that the effect of mCRP is persistent and sustained for at least 24 hours, regardless of the proinflammatory environment. In fact, mCRP exerted its action on human primary AF cells coming not only from patients with IVD degeneration, but also from healthy subjects. In accordance with this, we found that mCRP can be localized physiologically both in healthy NP and AF tissues and, in addition, mCRP presence seems to increase in the degenerative AF tissue compared to the healthy one. Our finding suggests that elevated locally produced levels of mCRP may also trigger sustained multigenic inflammatory responses in normal tissues not previously exposed to a proinflammatory micro-environment, as in the case of AF cells from degenerated IVD. Another relevant aspect is that the effects of mCRP were observed both in AF and NP cells, showing that mCRP has multiple cell targets. The last important insight of this work is that mCRP effects in AF cells are mediated by the NF-κβ signalling pathway. We proved that upon mCRP challenge, PI3K, ERK1/2, and NF-κβ p65 were phosphorylated. These mediators were activated in a similar manner both in healthy and degenerative AF disc cells, confirming that mCRP action does not depend on a previous inflammatory state.
Our results are in agreement with those obtained by other groups working with other cell types. For instance, it has been observed in osteoclasts that mCRP acts through the PI3K signalling pathway.60 Wang et al61 demonstrated that CRP increases IL-8 gene expression in human umbilical vein endothelial cells (HUVECs). Previous published results in cartilage showed the proinflammatory effect of mCRP in chondrocytes, clearly demonstrating that mCRP triggers a sustained proinflammatory and catabolic state in OA and healthy cartilage. These results also proved that NF-κβ has a role in mCRP signalling in cartilage.24 This experimental design and the results observed were similar to those obtained in IVD cells.
The limited access to primary degenerative and healthy human disc cells is a limitation of this study. Further studies involving a larger number of subjects would be needed to fully understand the role of mCRP in IVD degeneration pathophysiology.
In conclusion, our present findings provide, for the first time, evidence of the mCRP localization in healthy and degenerative IVD tissues, and a novel molecular basis for the sustained action of mCRP in human AF and NP cells. Taken together, these results suggest that mCRP exerts a lasting catabolic effect by increasing the expression of inflammatory mediators and proteolytic enzymes, being able to promote IVD cell breakdown and trigger inflammatory responses in healthy and degenerative AF cells and in the immortalized NP cell line. Altogether, these molecules can cooperate, resulting in the enhancement and perpetuation of the ECM-degrading processes at IVD level. In addition, mCRP by itself can trigger a severe increase in the expression of inflammatory mediators in healthy disc cells, suggesting that mCRP has a clear pathogenic role in disc disease. Our results indicate that this effect occurs both in AF and NP cells, and so affects the two main disc cellular compartments. Besides these findings, further studies on the pathophysiology of mCRP are needed to completely determine the role of mCRP in the pathogenesis and progression of IVD degeneration.
References
1. Hartvigsen J , Hancock MJ , Kongsted A , et al. What low back pain is and why we need to pay attention . Lancet . 2018 ; 391 ( 10137 ): 2356 – 2367 . Crossref PubMed Google Scholar
2. Andersson GB . Epidemiological features of chronic low-back pain . Lancet . 1999 ; 354 ( 9178 ): 581 – 585 . Crossref PubMed Google Scholar
3. Sampara P , Banala RR , Vemuri SK , Av GR , Gpv S . Understanding the molecular biology of intervertebral disc degeneration and potential gene therapy strategies for regeneration: a review . Gene Ther . 2018 ; 25 ( 2 ): 67 – 82 . Crossref PubMed Google Scholar
4. Francisco V , Pino J , González-Gay MÁ , et al. A new immunometabolic perspective of intervertebral disc degeneration . Nat Rev Rheumatol . 2022 ; 18 ( 1 ): 47 – 60 . Crossref PubMed Google Scholar
5. Ruiz-Fernández C , Francisco V , Pino J , et al. Molecular relationships among obesity, inflammation and intervertebral disc degeneration: Are adipokines the common link? Int J Mol Sci . 2019 ; 20 ( 8 ): 2030 . Crossref PubMed Google Scholar
6. Sakai D , Andersson GBJ . Stem cell therapy for intervertebral disc regeneration: obstacles and solutions . Internet . Nat Rev Rheumatol . 2015 ; 11 ( 4 ): 243 – 256 . Crossref PubMed Google Scholar
7. Freemont AJ , Peacock TE , Goupille P , Hoyland JA , O’Brien J , Jayson MI . Nerve ingrowth into diseased intervertebral disc in chronic back pain . Lancet . 1997 ; 350 ( 9072 ): 178 – 181 . Crossref PubMed Google Scholar
8. Carreon LY , Ito T , Yamada M , Uchiyama S , Takahashi HE . Neovascularization induced by anulus and its inhibition by cartilage endplate. Its role in disc absorption . Spine (Phila Pa 1976) . 1997 ; 22 ( 13 ): 1429 – 1434; discussion 1446-7 . Crossref PubMed Google Scholar
9. Liang Y , Xu K , Liu W , et al. Monomeric C-reactive protein level is associated with osteoarthritis . Exp Ther Med . 2022 ; 23 ( 4 ): 277 . Crossref PubMed Google Scholar
10. Zeller J , Bogner B , McFadyen JD , et al. Transitional changes in the structure of C-reactive protein create highly pro-inflammatory molecules: Therapeutic implications for cardiovascular diseases . Pharmacol Ther . 2022 ; 235 : 108165 . Crossref PubMed Google Scholar
11. Zha Z , Cheng Y , Cao L , et al. Monomeric CRP aggravates myocardial injury after myocardial infarction by polarizing the macrophage to pro-inflammatory phenotype through JNK signaling pathway . J Inflamm Res . 2021 ; 14 : 7053 – 7064 . Crossref PubMed Google Scholar
12. Fujita C , Sakurai Y , Yasuda Y , Takada Y , Huang CL , Fujita M . Anti-monomeric C-reactive protein antibody ameliorates arthritis and nephritis in mice . J Immunol . 2021 ; 207 ( 7 ): 1755 – 1762 . Crossref PubMed Google Scholar
13. Sproston NR , Ashworth JJ . Role of C-reactive protein at sites of inflammation and infection . Front Immunol . 2018 ; 9 : 754 . Crossref PubMed Google Scholar
14. Ciubotaru I , Potempa LA , Wander RC . Production of modified C-reactive protein in U937-derived macrophages . Exp Biol Med . 2005 ; 230 ( 10 ): 70 – 762 . Crossref PubMed Google Scholar
15. Le Gars L , Borderie D , Kaplan G , Berenbaum F . Systemic inflammatory response with plasma C-reactive protein elevation in disk-related lumbosciatic syndrome . Joint Bone Spine . 2000 ; 67 ( 5 ): 452 – 455 . PubMed Google Scholar
16. Hanada M , Takahashi M , Furuhashi H , Koyama H , Matsuyama Y . Elevated erythrocyte sedimentation rate and high-sensitivity C-reactive protein in osteoarthritis of the knee: relationship with clinical findings and radiographic severity . Ann Clin Biochem . 2016 ; 53 ( Pt 5 ): 548 – 553 . Crossref PubMed Google Scholar
17. Calabró P , Willerson JT , Yeh ETH . Inflammatory cytokines stimulated C-reactive protein production by human coronary artery smooth muscle cells . Circulation . 2003 ; 108 ( 16 ): 1930 – 1932 . Crossref PubMed Google Scholar
18. Devaraj S , Singh U , Jialal I . The evolving role of C-reactive protein in atherothrombosis . Internet . Clin Chem . 2009 ; 55 ( 2 ): 229 – 238 . Crossref PubMed Google Scholar
19. Pasceri V , Willerson JT , Yeh ETH . Direct proinflammatory effect of C-reactive protein on human endothelial cells . Circulation . 2000 ; 102 ( 18 ): 2165 – 2168 . Crossref PubMed Google Scholar
20. Calabro P , Chang DW , Willerson JT , Yeh ETH . Release of C-reactive protein in response to inflammatory cytokines by human adipocytes: linking obesity to vascular inflammation . J Am Coll Cardiol . 2005 ; 46 ( 6 ): 1112 – 1113 . Crossref PubMed Google Scholar
21. Wu Y , Potempa LA , El Kebir D , Filep JG . C-reactive protein and inflammation: conformational changes affect function . Biol Chem . 2015 ; 396 ( 11 ): 1181 – 1197 . Crossref PubMed Google Scholar
22. Khreiss T , József L , Potempa LA , Filep JG . Opposing effects of C-reactive protein isoforms on shear-induced neutrophil-platelet adhesion and neutrophil aggregation in whole blood . Circulation . 2004 ; 110 ( 17 ): 2713 – 2720 . Crossref PubMed Google Scholar
23. Du Clos TW . Pentraxins: structure, function, and role in inflammation . ISRN Inflamm . 2013 ; 2013 : 379040 . Crossref PubMed Google Scholar
24. Ruiz-Fernández C , Gonzalez-Rodríguez M , Francisco V , et al. Monomeric C reactive protein (mCRP) regulates inflammatory responses in human and mouse chondrocytes . Lab Invest . 2021 ; 101 ( 12 ): 1550 – 1560 . Crossref PubMed Google Scholar
25. Thiele JR , Habersberger J , Braig D , et al. Dissociation of pentameric to monomeric C-reactive protein localizes and aggravates inflammation: in vivo proof of a powerful proinflammatory mechanism and a new anti-inflammatory strategy . Circulation . 2014 ; 130 ( 1 ): 35 – 50 . Crossref PubMed Google Scholar
26. Mok JM , Pekmezci M , Piper SL , et al. Use of C-reactive protein after spinal surgery: comparison with erythrocyte sedimentation rate as predictor of early postoperative infectious complications . Spine (Phila Pa 1976) . 2008 ; 33 ( 4 ): 415 – 421 . Crossref PubMed Google Scholar
27. van Gerven C , Eid K , Krüger T , et al. Serum C-reactive protein and WBC count in conservatively and operatively managed bacterial spondylodiscitis . J Orthop Surg (Hong Kong) . 2021 ; 29 ( 1 ): 2309499020968296 . Crossref PubMed Google Scholar
28. Potempa LA , Yao Z-Y , Ji S-R , Filep JG , Wu Y . Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification . Biophys Rep . 2015 ; 1 : 18 – 33 . Crossref PubMed Google Scholar
29. Sakai D , Mochida J , Yamamoto Y , et al. Immortalization of human nucleus pulposus cells by a recombinant SV40 adenovirus vector: establishment of a novel cell line for the study of human nucleus pulposus cells . Spine (Phila Pa 1976) . 2004 ; 29 ( 14 ): 1515 – 1523 . Crossref PubMed Google Scholar
30. World Medical Association . World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects . JAMA . 2013 ; 310 ( 20 ): 2191 – 2194 . PubMed Google Scholar
31. Otero M , Favero M , Dragomir C , et al. Human chondrocyte cultures as models of cartilage-specific gene regulation . Methods Mol Biol . 2012 ; 806 : 301 – 336 . Crossref PubMed Google Scholar
32. Scotece M , Conde J , Abella V , et al. Oleocanthal inhibits catabolic and inflammatory mediators in LPS-activated human primary osteoarthritis (OA) chondrocytes through MAPKs/NF-κB pathways . Cell Physiol Biochem . 2018 ; 49 ( 6 ): 2414 – 2426 . Crossref PubMed Google Scholar
33. Lago R , Gomez R , Otero M , et al. A new player in cartilage homeostasis: adiponectin induces nitric oxide synthase type II and pro-inflammatory cytokines in chondrocytes . Osteoarthritis Cartilage . 2008 ; 16 ( 9 ): 1101 – 1109 . Crossref PubMed Google Scholar
34. Ethemoğlu KB , Erkoç YS . Is there any relationship between cervical disc herniation and blood inflammatory response? Cureus . 2020 ; 12 ( 8 ): e10161 . Crossref PubMed Google Scholar
35. Thiele JR , Zeller J , Bannasch H , Stark GB , Peter K , Eisenhardt SU . Targeting C-reactive protein in inflammatory disease by preventing conformational changes . Mediators Inflamm . 2015 ; 2015 : 372432 . Crossref PubMed Google Scholar
36. Khreiss T , József L , Hossain S , Chan JSD , Potempa LA , Filep JG . Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils . J Biol Chem . 2002 ; 277 ( 43 ): 40775 – 40781 . Crossref PubMed Google Scholar
37. Uvodich ME , Dugdale EM , Osmon DR , Pagnano MW , Berry DJ , Abdel MP . The effectiveness of laboratory tests to predict early postoperative periprosthetic infection after total knee arthroplasty . Bone Joint J . 2021 ; 103-B ( 6 Supple A ): 177 – 184 . Crossref PubMed Google Scholar
38. de Souza Grava AL , Ferrari LF , Defino HLA . Cytokine inhibition and time-related influence of inflammatory stimuli on the hyperalgesia induced by the nucleus pulposus . Eur Spine J . 2012 ; 21 ( 3 ): 537 – 545 . Crossref PubMed Google Scholar
39. Stürmer T , Raum E , Buchner M , et al. Pain and high sensitivity C reactive protein in patients with chronic low back pain and acute sciatic pain . Ann Rheum Dis . 2005 ; 64 ( 6 ): 921 – 925 . Crossref PubMed Google Scholar
40. Sugimori K , Kawaguchi Y , Morita M , Kitajima I , Kimura T . High-sensitivity analysis of serum C-reactive protein in young patients with lumbar disc herniation . J Bone Joint Surg Br . 2003 ; 85-B ( 8 ): 1151 – 1154 . Crossref PubMed Google Scholar
41. Lowe GDO , Pepys MB . C-reactive protein and cardiovascular disease: weighing the evidence . Curr Atheroscler Rep . 2006 ; 8 ( 5 ): 421 – 428 . Crossref PubMed Google Scholar
42. Lloyd-Jones DM , Liu K , Tian L , Greenland P . Narrative review: Assessment of C-reactive protein in risk prediction for cardiovascular disease . Internet . Ann Intern Med . 2006 ; 145 ( 1 ): 35 – 42 . Crossref PubMed Google Scholar
43. Sattar N , Hingorani AD . C-reactive protein and prognosis in diabetes: getting to the heart of the matter . Diabetes . 2009 ; 58 ( 4 ): 798 – 799 . Crossref PubMed Google Scholar
44. Elliott P , Chambers JC , Zhang W , et al. Genetic Loci associated with C-reactive protein levels and risk of coronary heart disease . JAMA . 2009 ; 302 ( 1 ): 37 – 48 . Crossref PubMed Google Scholar
45. Yousuf O , Mohanty BD , Martin SS , et al. High-sensitivity C-reactive protein and cardiovascular disease: A resolute belief or an elusive link? J Am Coll Cardiol . 2013 ; 62 ( 5 ): 397 – 408 . Google Scholar
46. Chang YH , Hwu DW , Kao WP , Lee YJ . Benefits of rosuvastatin in cardiovascular protection remain unclear after HOPE-3 . Rev Diabet Stud . 2016 ; 13 ( 4 ): 212 – 214 . Crossref PubMed Google Scholar
47. Antonelli M , Kushner I . It’s time to redefine inflammation . FASEB J . 2017 ; 31 ( 5 ): 1787 – 1791 . Google Scholar
48. No authors listed . Review Criteria for Assessment of C Reactive Protein (CRP), High Sensitivity C-Reactive Protein (hsCRP) and Cardiac C-Reactive Assays - Guidance for Industry and FDA Staff . U.S. Food and Drug Administration (FDA) . 2005 . https://www.fda.gov/regulatory-information/search-fda-guidance-documents/review-criteria-assessment-c-reactive-protein-crp-high-sensitivity-c-reactive-protein-hscrp-and ( date last accessed 15 February 2023 ). Google Scholar
49. Risbud MV , Shapiro IM . Role of cytokines in intervertebral disc degeneration: pain and disc content . Nat Rev Rheumatol . 2014 ; 10 ( 1 ): 44 – 56 . Crossref PubMed Google Scholar
50. Kang JD , Georgescu HI , McIntyre-Larkin L , Stefanovic-Racic M , Donaldson WF , Evans CH . Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 . Spine (Phila Pa 1976) . 1996 ; 21 ( 3 ): 271 – 277 . Crossref PubMed Google Scholar
51. Guo Z , Qiu C , Mecca C , et al. Elevated lymphotoxin-α (TNFβ) is associated with intervertebral disc degeneration . BMC Musculoskelet Disord . 2021 ; 22 ( 1 ): 77 . Crossref PubMed Google Scholar
52. Jia Z-K , Li H-Y , Liang Y-L , Potempa LA , Ji S-R , Wu Y . Monomeric C-reactive protein binds and neutralizes receptor activator of NF-κB ligand-induced osteoclast differentiation . Front Immunol . 2018 ; 9 : 234 . Crossref PubMed Google Scholar
53. Clouet J , Grimandi G , Pot-Vaucel M , et al. Identification of phenotypic discriminating markers for intervertebral disc cells and articular chondrocytes . Rheumatology (Oxford) . 2009 ; 48 ( 11 ): 1447 – 1450 . Crossref PubMed Google Scholar
54. Sadowska A , Touli E , Hitzl W , et al. Inflammaging in cervical and lumbar degenerated intervertebral discs: analysis of proinflammatory cytokine and TRP channel expression . Eur Spine J . 2018 ; 27 ( 3 ): 564 – 577 . Crossref PubMed Google Scholar
55. Francisco V , Pérez T , Pino J , et al. Biomechanics, obesity, and osteoarthritis. The role of adipokines: When the levee breaks . J Orthop Res . 2018 ; 36 : 594 – 604 . Crossref PubMed Google Scholar
56. Francisco V , Ruiz-Fernández C , Pino J , et al. Adipokines: linking metabolic syndrome, the immune system, and arthritic diseases . Biochem Pharmacol . 2019 ; 165 : 196 – 206 . Crossref PubMed Google Scholar
57. Abella V , Scotece M , Conde J , et al. Adipokines, metabolic syndrome and rheumatic diseases . J Immunol Res . 2014 ; 2014 : 343746 . Crossref PubMed Google Scholar
58. Khreiss T , József L , Potempa LA , Filep JG . Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells . Circulation . 2004 ; 109 ( 16 ): 2016 – 2022 . Crossref PubMed Google Scholar
59. Khreiss T , József L , Potempa LA , Filep JG . Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils . Circ Res . 2005 ; 97 ( 7 ): 690 – 697 . Crossref PubMed Google Scholar
60. Boras E , Slevin M , Alexander MY , et al. Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway . Cytokine . 2014 ; 69 ( 2 ): 165 – 179 . Crossref PubMed Google Scholar
61. Wang Q , Zhu X , Xu Q , Ding X , Chen YE , Song Q . Effect of C-reactive protein on gene expression in vascular endothelial cells . Am J Physiol Heart Circ Physiol . 2005 ; 288 ( 4 ): H1539 – 45 . Crossref PubMed Google Scholar
Author contributions
C. Ruiz-Fernández: Investigation, Methodology, Formal analysis, Data curation, Writing – original draft, Writing – review & editing.
D. Ait Eldjoudi: Investigation, Data curation.
M. González-Rodríguez: Investigation, Data curation.
A. Cordero Barreal: Investigation, Data curation, Writing – review & editing.
Y. Farrag: Investigation, Formal analysis, Writing – review & editing.
L. García-Caballero: Investigation, Resources, Writing – review & editing.
F. Lago: Formal analysis, Data curation, Writing – review & editing.
A. Mobasheri: Data analysis, Writing – review & editing.
D. Sakai: Data analysis, Writing – review & editing.
J. Pino: Conceptualization, Supervision, Data curation, Writing – original draft, Writing – review & editing.
O. Gualillo: Conceptualization, Supervision, Data curation, Writing – original draft, Writing – review & editing.
Funding statement
The authors disclose receipt of the following financial or material support for the research, authorship, and/or publication of this article: funding from the ISCIII (Instituto de Salud Carlos III) and The European Regional Development Fund (ERDF- FEDER) (grant PI20/00902).
ICMJE COI statement
The authors declare no competing interests.
Acknowledgements
The authors kindly acknowledge Professor Lawrence Potempa and Dr Ibraheem Rajab (Roosevelt University, College of Pharmacy, USA) for providing them with the recombinant monomeric CRP.
Ethical review statement
The current study was approved by the Galician Ethics Committee (Comité Autonómico de Ética da Investigación de Galicia, Xunta de Galicia, Consellería de Sanidade. Código de Registro: 2017/279), and was conducted in accordance with the Declaration of Helsinki. Signed written informed consents were obtained from each donor.
Open access funding
The authors report that they received open access funding for their manuscript by grant PI20/00902 funded by ISCIII (Instituto de Salud Carlos III) and The European Regional Development Fund (ERDF- FEDER).
Follow O. Gualillo @neiridlab
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