Abstract
BACKGROUND CONTEXT
Inflammation has been associated with a number of pathological conditions including
intervertebral disc (IVD) degeneration, increased risks of low back pain and other
spinal diseases. Downregulating disc inflammation may be a strategy to reduce degeneration
and more importantly back pain. Interleukin (IL)-4 was first discovered as a T-cell
secreted factor that enhanced the proliferation of anti-IgM stimulated B cells and
is now known as a cytokine that can stimulate cell proliferation and differentiation,
tissue regeneration and neurological functions. IL-4 has been shown to be effective
in inhibiting inflammatory pathways in chondrocytes. Immunohistochemical studies have
shown that disc tissues are immunopositive for IL-4 receptor α (IL-4Rα) and IL-4.
Yet, the roles of IL-4 and IL-4R in disc biology remain unknown.
PURPOSE
The purpose of this study is to understand the roles of IL-4 and IL-4Rα in IVDs and
to determine if IL-4 can function to inhibit inflammation in IVD cells.
STUDY DESIGN/SETTING
In vitro experiment.
METHODS
Deidentified patient IVD tissues were collected after surgery under the Orthopedic
Information, Tissue and Implant Repository (ORA L00011021). IVD cells were isolated
and cultured in monolayer. IL-4R protein expression was analyzed using immunocytochemistry.
To test if the IL-4R was responsive to its ligand, signal transducer and activator
of transcription 6 (STAT6) phosphorylation was analyzed on cell lysates of IVD cells
treated with recombinant human IL-4 for 30 minutes using enzyme linked immunosorbent
assay kit. Gene expression analysis of IL-4 up- and downregulated genes were analyzed
using real-time RT-PCR. Anti-inflammatory effects of IL-4 were determined by cotreating
disc cells with lipopolysaccharide (LPS) and IL-4 and measuring gene expression and
protein release of inflammatory markers, IL-6 and IL-8. The significance of differences
among means of data on gene expression and protein analyses were analyzed by one-way
analysis of variance or student t test. Differences were considered significant when the p value was below 0.05.
RESULTS
Immunocytochemistry staining for IL-4Rα in primary IVD cells (n=8) showed the majority
of immunopositive staining was intracellular. After IVD cells (n=3–7) were treated
with different concentrations of recombinant human IL-4 (0.1–100 ng/mL) for 30 minutes,
phospho-STAT6 levels significantly increased by two- to four-fold at all concentrations
tested compared with untreated cells. Gene expression of IL-4Rα and IL-6 increased
significantly in cells undergoing IL-4 treatment for 24 hours compared with control
treated IVD cells (n=5–10). LPS stimulated inflammatory gene expression of interferon
(IFN)β, IL-12, IL-6, and IL-8 were downregulated significantly in the presence of
IL-4 (n=7). Lastly, protein release of IL-6 and IL-8 were reduced significantly in
cells treated with IL-4 and LPS compared with those treated with LPS alone (n=7).
CONCLUSIONS
This study was the first to explore the function of IL-4 and IL-4R in IVD cells. Immunocytochemistry
studies confirmed that the majority of cells isolated from patient IVDs expressed
IL-4Rα at the protein level. Also, IVD cells can respond to IL-4 by up-regulating
IL-4Rα and IL-6 genes and inhibiting inflammatory genes and proteins induced by LPS.
Further studies to test the anti-inflammatory effects of IL-4 in the IVD would be
needed in animal models.
CLINICAL RELEVANCE
Biological therapies which include intradiscal delivery of cells, anti-inflammatories
or growth factors are being investigated to treat disc degeneration and back pain
in animal models and in the clinic. Based on our findings that IL-4 has anti-inflammatory
effects on IVD cells, the results of this study suggest including recombinant IL-4
delivery into the intervertebral disc may be a beneficial therapeutic strategy to
treat patients with back pain by reducing disc inflammation.
Keywords
To read this article in full you will need to make a payment
Purchase one-time access:
Academic and PersonalCorporate R&D ProfessionalsOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to The Spine JournalAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
- Low back pain in relation to lumbar disc degeneration.Spine (Phila Pa.1976). 2000; 25: 487-492
- Role of cytokines in intervertebral disc degeneration: pain and disc content.Nat Rev Rheumatol. 2014; 10: 44-56
- Inflammation in intervertebral disc degeneration and regeneration.J R Soc Interface. 2015; 1220150429
- Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs: a study in surgical specimen and autopsy controls.Spine (Phila Pa.1976). 2005; 30 (53; discussion 54): 44
- Herniated and spondylotic intervertebral discs of the human cervical spine: histological and immunohistological findings in 500 en bloc surgical samples. Laboratory investigation.J Neurosurg Spine. 2008; 9: 285-295
- Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile.Arthritis Res Ther. 2007; 9: R77
- Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2.Spine. 1996; 21: 271-277
- Inflammatory cytokines in the herniated disc of the lumbar spine.Spine. 1996; 21: 218-224
- Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators.J Bone Joint Surg Br. 2002; 84: 196-201
- mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs.Spine (Phila Pa.1976). 2002; 27: 911-917
- Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues.Arthritis Rheum. 2010; 62: 1974-1982
- Spontaneous production of monocyte chemoattractant protein-1 and interleukin-8 by the human lumbar intervertebral disc.Spine (Phila Pa.1976). 2002; 27: 1402-1407
- Tumor necrosis factor alpha- and interleukin-1beta-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1.Arthritis Rheum. 2013; 65: 832-842
- Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-beta and TNF-alpha in 3D culture.Exp Mol Pathol. 2014; 96: 133-138
- Chemokine receptor antagonists can inhibit macrophage migration induced by annulus fibrosus and nucleus pulposus cells.in: International Society for the Advancement of Spine Surgery Annual Meeting. 2015
- Identification of a T cell-derived b cell growth factor distinct from interleukin 2.J Exp Med. 1982; 155: 914-923
- History of Interleukin-4.Cytokine. 2015; 75: 3-7
- Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4.J Immunol. 1988; 140: 1548-1554
- Influence of pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammatory (IL-4) cytokines on chondrocyte function.Osteoarthritis Cartilage. 2003; 11: 681-687
- Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes.Biochem Biophys Res Commun. 2006; 339: 241-247
- Expression of canine interleukin-4 in canine chondrocytes inhibits inflammatory cascade through STAT6.Cytokine. 2008; 44: 179-184
- Human osteoarthritic cartilage shows reduced in vivo expression of IL-4, a chondroprotective cytokine that differentially modulates IL-1beta-stimulated production of chemokines and matrix-degrading enzymes in vitro.PLoS One. 2014; 9: e96925
- Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis.J Immunol. 1993; 151: 4344-4351
- Intra-articular injection of interleukin-4 decreases nitric oxide production by chondrocytes and ameliorates subsequent destruction of cartilage in instability-induced osteoarthritis in rat knee joints.Osteoarthritis Cartilage. 2008; 16: 764-771
- Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.J Immunol. 1999; 163: 4546-4556
- Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium.Mol Ther. 2000; 2: 147-152
- Local overexpression of adeno-viral IL-4 protects cartilage from metallo proteinase-induced destruction during immune complex-mediated arthritis by preventing activation of pro-MMPs.Osteoarthritis Cartilage. 2002; 10: 234-243
- Targeted IL-4 therapy synergizes with dexamethasone to induce a state of tolerance by promoting Treg cells and macrophages in mice with arthritis.Eur J Immunol. 2016; 46: 1246-1257
- The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration.Arthritis Res Ther. 2011; 13: R8
- Expression of high affinity receptors for murine interleukin 4 (BSF-1) on hemopoietic and nonhemopoietic cells.J Immunol. 1988; 140: 456-464
- Interleukin-4 (IL-4) receptor expression on human T cells is affected by different intracellular signaling pathways and by IL-4 at transcriptional and posttranscriptional level.Blood. 1992; 80: 2721-2728
- Divergent effects of interleukin-4 and interferon-gamma on macrophage-derived chemokine production: an amplification circuit of polarized T helper 2 responses.Blood. 1998; 92: 2668-2671
- Interleukin-13 stimulates interleukin-6 production by human keratinocytes. Similarity with interleukin-4.FEBS Lett. 1994; 343: 32-36
- IL-4 inhibits the expression of IL-8 from stimulated human monocytes.J Immunol. 1990; 145: 1435-1439
- Tissue-specific regulation of IL-6 production by IL-4. Differential effects of IL-4 on nuclear factor-kappa B activity in monocytes and fibroblasts.J Immunol. 1993; 151: 5603-5612
- Stimulatory and inhibitory effects of interleukin (IL)-4 and IL-13 on the production of cytokines by human peripheral blood mononuclear cells: priming for IL-12 and tumor necrosis factor alpha production.J Exp Med. 1995; 181: 537-546
- Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines.J Immunol. 1996; 156: 2591-2598
- IL-4 inhibits the production of TNF-alpha and IL-12 by STAT6-dependent and -independent mechanisms.J Immunol. 1999; 162: 5224-5229
- Interleukin 6: from bench to bedside.Nat Clin Pract Rheumatol. 2006; 2: 619-626
- Physiological effects of modulating the interleukin-6 axis.Rheumatology (Oxford). 2018; 57: ii43-ii50
- IL4 induces IL6-producing M2 macrophages associated to inhibition of neuroinflammation in vitro and in vivo.J Neuroinflammation. 2016; 13 (016-0596-5): 139
- Requirement for dual signals by anti-CD40 and IL-4 for the induction of nuclear factor-kappa B, IL-6, and IgE in human B lymphocytes.J Immunol. 1998; 161: 1738-1742
- Interleukin-4 (IL-4) inhibits secretion of IL-1 beta, tumor necrosis factor alpha, and IL-6 by human monocytes.Blood. 1990; 76: 1392-1397
- Suppression of metalloproteinase biosynthesis in human alveolar macrophages by interleukin-4.J Clin Invest. 1992; 90: 382-388
- Coordinated antiinflammatory effects of interleukin 4: interleukin 4 suppresses interleukin 1 production but up-regulates gene expression and synthesis of interleukin 1 receptor antagonist.Proc Natl Acad Sci USA. 1992; 89: 4076-4080
- Interferons: success in anti-viral immunotherapy.Cytokine Growth Factor Rev. 2014; 25: 369-376
- Current perspectives on interferon Beta-1b for the treatment of multiple sclerosis.Adv Ther. 2014; 31: 915-931
- IL-2: the first effective immunotherapy for human cancer.J Immunol. 2014; 192: 5451-5458
- Filgrastim (r-metHuG-CSF): the first 10 years.Blood. 1996; 88: 1907-1929
- Clinical applications of granulocyte colony-stimulating factor: an update and summary.Ann Hematol. 2003; 82: 207-213
- An update on sargramostim for treatment of acute radiation syndrome.Drugs Today (Barc). 2018; 54: 679-693
Yeh L, Augustine AJ, Sheldon A. Use of IL-4 for inhibition of the breakdown of articular cartilage and other tissues, United States patent US-5679338-A, 1997.
- Dupilumab: first global approval.Drugs. 2017; 77: 1115-1121
- 2009 ISSLS Prize Winner: does discography cause accelerated progression of degeneration changes in the lumbar disc: a ten-year matched cohort study.Spine. 2009; 34: 2338-2345
Article Info
Publication History
Published online: June 29, 2019
Accepted:
June 28,
2019
Received in revised form:
June 27,
2019
Received:
November 28,
2018
Footnotes
FDA device/drug status: Not applicable.
Author disclosures: HK: Nothing to disclose. DW: Nothing to disclose. MS: Nothing to disclose. HSA: Royalties: U&I Inc (B); Stock Ownership: Spinal Kinetics (A), Medyssey Inc (C); Consulting: Bioventus Inc (B); Endowments: Rush University Medical Center (D); Research Support (Investigator Salary, Staff/Materials): ChemoCentryx (Material support [CCR inhibitors]); Grants: Aircast Foundation (PI) (E), Pritzker Pucker Family Foundation (PI) (F), Cervical Spine Research Society (Co-I) (F), AO Foundation (PI) (F), Yuhan Corp. (PI) (F), Spinalcyte, LLC (PI) (F); Fellowship Support: AO Spine (F). AC: Grants: Cervical Spine Research Society (PI) (F), Aircast Foundation (Co-I) (E), Pritzker Pucker Family Foundation (Co-I) (F), AO Foundation (Co-I) (F), North American Spine Society (Co-I) (D), Yuhan Corp. (Co-I) (F), Spinalcyte, LLC (Co-I) (F).
Identification
Copyright
© 2019 Elsevier Inc. All rights reserved.
