Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.
METHODS
Laboratory study.


OBJECTIVE
To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP.


BACKGROUND
SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells.


METHODS
Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP.


RESULTS
We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells.


CONCLUSIONS
NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways.


METHODS
4.
*Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA †Department of Orthopaedic Surgery, Rothman Institute; and ‡Department of Orthopedics, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
Christopher K
CK
Kepler
Dessislava Z
DZ
Markova
John D
JD
Koerner
Joseph
J
Mendelis
Chiu-Ming
CM
Chen
Alexander R
AR
Vaccaro
Makarand V
MV
Risbud
Todd J
TJ
Albert
D Greg
DG
Anderson
D000328:Adult; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D016209:Interleukin-8 / Q000235:genetics; D007378:Interleukins / Q000235:genetics*; D007403:Intervertebral Disc / Q000166:cytology*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D008875:Middle Aged; D064729:Neurokinin-1 Receptor Antagonists / Q000494:pharmacology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000235:genetics; D018041:Receptors, Neurokinin-2 / Q000235:genetics; D018042:Receptors, Neurokinin-3 / Q000235:genetics; D013373:Substance P / Q000494:pharmacology*; D051996:Transcription Factor RelA / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D007378:Interleukins; D064729:Neurokinin-1 Receptor Antagonists; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1; D018041:Receptors, Neurokinin-2; D018042:Receptors, Neurokinin-3; D051996:Transcription Factor RelA; D013373:Substance P; D048051:p38 Mitogen-Activated Protein Kinases