J Manipulative Physiol Ther. 2011 (Oct); 34 (8): 498–505 ~ FULL TEXT
Julita A. Teodorczyk-Injeyan, PhD, John J. Triano, DC, PhD, Marion McGregor, DC, PhD,
Linda Woodhouse, PT, PhD, H. Stephen Injeyan, PhD, DC
Graduate Education and Research Programs,
Canadian Memorial Chiropractic College,
Toronto, Ontario, Canada.
OBJECTIVE: This study investigated whether the production of inflammatory mediators and chemotactic cytokines (chemokines) is altered in patients with chronic and recurrent neck pain (NP).
METHODS: Cross-sectional data evaluating blood and serum samples were obtained from 27 NP patients and 13 asymptomatic (control) subjects recruited from a chiropractic outpatient clinic. Cell cultures were activated by lipopolysaccharide (LPS) and phytoheamagglutinin for 24 to 48 hours. The levels of tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1, also known as CCL2 (CCL2/MCP-1), and macrophage inflammatory protein 1α or CCL3 (CCL3/MIP-1α) were determined by specific immunoassays. Serum levels of nitric oxide metabolites were evaluated simultaneously, in vanadium III-reduced samples, by Griess reaction.
RESULTS: Low levels of constitutive (spontaneous) TNF-α production were present in 7 of the 27 cultures from patients with NP. Both LPS-induced TNF-α production and inducer (LPS/phytoheamagglutin)-stimulated production of CCL2 were significantly elevated (P = .00) in patients compared with controls. In patients, the constitutive synthesis of CCL3 occurred significantly more frequently (P = .00) and ranged from 30 to more than 2000 pg/mL. Finally, serum levels of nitric oxide were significantly elevated (P = .00) in NP patients.
CONCLUSIONS: Production of inflammatory mediators was consistently elevated in NP patients in this study, both in vitro and in vivo, and activation of inflammatory pathways was accompanied by up-regulation of CC chemokine synthesis. This suggests that, in NP patients, CC chemokines may be involved in regulation of local inflammatory response through recruitment of immune cells to the inflamed tissue and exert pronociceptive effects.
Key Indexing Terms: Neck Pain, Inflammation, Cytokines, Chemokines
From the FULL TEXT Article:
The role of inflammatory mediators in the genesis of persistent pain and hyperalgesia has been recently explored in different clinical and experimental models. [1, 2] In certain clinical conditions associated with chronic pain, proinflammatory cytokine levels appear to correlate with pain intensity.  However, the involvement of chemical mediators of inflammation in the pathology of chronic and/or acute spinal pain remains unclear despite localization of inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)- 1β, and IL-6 both in intervertebral disc specimens [4-6] and in facet joint tissues of degenerating lumbar spine. [7, 8]
The cause of neck pain (NP) is generally considered to be multifactorial and nonspecific.  Current medical opinion favors a prevalence of cervical facet joint pain in patients presenting with chronic NP. [10, 11] Evidence from animal studies pointing to facet involvement also exists. In a rodent model, Lee et al  have observed that tensile facet injury across the C6/C7 joint produces behavioral sensitivity suggestive of pain. Recently, a correlation of cervical and lumbar facet joint pain has been documented in a large clinical study,  and lumbar facet joint pain has been shown to be associated with the synthesis of inflammatory cytokines.  This may suggest that analogous inflammatory mediators may also be involved in the etiology of NP. Immunohistologic evidence of localized production of TNF-α in herniated and spondylotic intervertebral discs of the cervical spine  and increased levels of inflammatory cytokines (IL-1 and IL-6) in the cerebrospinal fluid from patients with cervical myelopathy  may also provide support for consideration of the involvement of inflammatory molecules in NP.
The initiation and continuation of the inflammatory response require a sustained recruitment and migration of immune and inflammatory cells from the circulation to inflamed tissues. This process is generated and mediated by a group of small chemotactic cytokines (chemokines), which form, based on the presence and position of the first cysteine (C) residues, 4 distinctive families.  Members of CC family, characterized by the presence of 2 adjacent cysteines, attract leukocytes to the site of injury by activating specific G-protein–coupled receptors on the leukocyte surface and act in concert with other cytokines to induce tissue infiltration and enrich the circulating pool of a given leukocyte.  In patients with inflammatory conditions, augmented production of CC chemokines has been reported to correlate with the severity of disease  and analgesia. 
The present study was designed to explore whether up-regulation of inflammatory mediator pathways can be demonstrated in patients with chronic and recurrent NP. To determine the state of activation of circulating peripheral blood mononuclear cells and the likelihood of their selected recruitment toward the site of inflammation, the in vitro production of a key proinflammatory cytokine, TNF-α, and the levels of synthesis of 2 chemokines, monocyte chemotactic protein 1, also known as CCL2, (MCP-1/CCL2) and macrophage inflammatory protein 1α, or CCL3, (MIP-1α/CCL3), respectively, were investigated. Because inflammatory and chemotactic cytokines are strong inducers of endogenous nitric oxide (NO) production and NO plays a role in mediation of peripheral and chronic nociception including spinal pain, [3, 20, 21] serum levels of NO were also examined.
The purpose of the present study was to investigate if NP is associated with a local inflammatory response that is accompanied by the release of quantifiable soluble mediators. The results support the hypothesis that NP syndrome is associated with activation of inflammatory cytokine pathways. Table 2 presents the summary of these results and depicts an inflammatory profile of the studied NP patients. Patients with NP, enrolled in the current study, demonstrated significantly elevated levels of systemic (in vivo) and/or in vitro–induced production of biomarkers inherent in physiologic responses involving inflammatory cells. Of particular interest is the observation of augmented production of the chemotactic cytokines, CCL2 and CCL3 (Fig 2, Fig 3, Table 2). The increased capacity for synthesis and release of chemokines is invariably associated with inflammation of tissues or organs,  and the principal stimuli enticing their synthesis are inflammatory, macrophage-derived cytokines such as TNF-α and IL-1.18 Although we were not able to observe increases in the level of systemic TNF-α in patients sera, the same subjects showed significant elevation of its production in vitro (Fig 1, Table 2). This is in concordance with observations that serum cytokine levels seldom correspond to the extent of synthesis and action of proinflammatory mediators produced in situ and accumulate predominantly in target tissues. [28, 29] On the other hand, sustainment of target tissue inflammation entails continuous production of such mediators delivered by infiltrating inflammatory cells, which migrate to the inflamed tissues from the periphery, recruited by chemokines. The investigative model used in our studies included functional assessment of the capacity for in vitro synthesis/release of inflammatory mediators by peripheral leukocytes obtained from NP patients. This approach allowed us to detect pathophysiologic changes in the state of activation of circulating inflammatory cells. Such changes might have not been discerned through studies restricted to scrutiny of patients' sera.
Chemokine system and the G-protein–coupled chemokine receptors provide essential guidance for interstitial leukocyte migration and trafficking.  Similarly, cytokines such as TNF-α can transmit diverse signals to different subsets of leukocytes and alter their adhesiveness and directional movement  Recently, Xu et al  reported that mechanisms controlling monocyte trafficking through the peripheral blood and lymphoid tissues are altered during local inflammation but the effect is systemic in maintaining inflammatory cells within the circulation. This observation may explain increases in the level of constitutive secretion of CC chemokines in cultures of unstimulated WB leukocytes as well as augmented synthesis of proinflammatory mediators (TNF-α, CCL2) in inducer-exposed preparations (Table 2).
The cellular origin of CCL-2 and CCL-3 in the studied patients remains unclear. As discussed above, under pathologic conditions, most chemokines are released by inflamed tissues and by infiltrating leukocytes. However, MCP-1 (CCL2) and inflammatory cytokines may be released also by intervertebral disc cells both in an experimental model6 as well as in cultures of human intervertebral disc cells.  However, in the present study, patients with evidence of radicular pain suggesting disc protrusion or stenosis were excluded. Based on the age range overlap between the patients and controls, it is unlikely that degenerative intervertebral disc tissue contributed to the observed elevation in the level of systemic CCL2. On the other hand, the recruited patients reported localized neck muscle pain associated with tenderness at palpation, stiffness, and constant muscle fatigue consistent with mechanical NP.  Human myoblasts and fibroblasts can spontaneously produce a variety of cytokines and chemokines; the transcription and expression of these mediators are strongly up-regulated in inflammatory conditions.  Strain-induced changes of cytokine secretion in an in vitro model of cultured fibroblasts have been reported,  suggesting a role of fibroblasts in the etiology of inflammatory change in conditions such as myofascial pain. Thus, neck muscles and contiguous connective tissues may also produce and release inflammatory and chemotactic cytokines involved in recruitment and activation of peripheral blood cells to the site of inflammation. Increased numbers of CC chemokine- and chemokine receptor–expressing peripheral blood mononuclear cells were reported by Kivioja et al  in patients with whiplash injuries. Although the capacity for chemokine release was not investigated in that study, their observations suggest that a local (whiplash-associated) soft tissue injury suffice for systemic up-regulation of chemokine and chemokine receptor expression.
Physiologically, the production of inflammatory cytokines and chemokines is regulated, positively or negatively, by other mediators including prostaglandins and NO. The present report demonstrates that, in NP patients, systemic NO production, assessed through the measurement of levels of the total serum nitrogen intermediates, (NO2)– and (NO3)–, is significantly elevated (Fig 3B). Considering the role of NO in the induction and resolution of pain and inflammation,  expression of high levels of its production may be reflective of a strong pathophysiologic response involving cells of the immune, nervous, and musculoskeletal systems. Nitric oxide is released via inducible NO synthase, which, in contrast to other isoforms of this enzyme, is expressed only in inflamed tissues and when leukocyte recruitment is occurring at a rapid rate. [38, 39] Experimental and clinical studies are in agreement that NO plays a major role in the induction of proinflammatory mediator production as well as in modulation of nociceptive signaling. [3, 40] The modulatory effect of NO on the mechanical responsiveness of nociceptors depends on the baseline level of neuronal excitability.  Both chemokines and inflammatory cytokines are involved in the generation of hyperexcitable sensory neurons and thereby participate in neural signaling associated with pain states.  Furthermore, chemokines, expressed by multiple cell types within the nervous system, may produce receptor-mediated nociceptive effects through desensitization of neural opioid receptors.  In this regard, CCL2 appears to be particularly important because its overexpression or administration induces manifestation of pain sensitivity in experimental models. [42, 43] These observations and results of our investigation suggest that the combined effects of inflammatory cytokine(s)-CC chemokine sensitization of neurons and enhancement of NO signaling could contribute to the genesis of NP in the studied patients. This concept gains further support from the observation of elevated levels of nitric metabolites in sera from patients with cervicogenic headache noted at the onset of headache but not during pain-free periods. 
To our knowledge, this report is the first to demonstrate activation of proinflammatory mediator pathways in patients experiencing NP. Further elucidation of the etiologic mechanisms of mechanical NP may furnish the necessary basic data to justify investigative studies of the effects of various therapeutic modalities in the treatment of this clinical condition.
The limitations of this study principally pertain to the patient population selected for the study. Patients with chronic and recurrent NP were recruited without strictly controlling for several key covariants that may influence the results including the etiology of injury at onset, the underlying pathophysiology responsible for the generation of pain, the duration of pain, and the psychological state of the participants. In addition, the intensity of pain and functional limitations varied among participants. This study made no attempt to explore possible associations between biomarker levels relative to pain intensity or disability scores because this would have required a larger sample size.
The present pilot study was an extension derived from a larger clinical trial investigating the effects of a physical modality on NP patients. The results reported herein encourage us to initiate a prospective study to address the limitations mentioned above and prepare groundwork for clinical trials examining the effects of different treatment interventions on chronic NP using changes in biomarker synthesis/secretion as outcomes.
The patients with chronic and recurrent NP in this study demonstrated elevated levels of inflammatory biomarkers production both in vitro and in vivo accompanied by up-regulation of CC chemokine synthesis. Thus, in NP, CC chemokines may be involved in the mediation of the local inflammatory response through the recruitment of immune cells to the inflamed tissues. Furthermore, in concert with NO, they may exert pronociceptive effects.
Production of chemotactic cytokines (chemokines), known to be induced
by proinflammatory cytokines, is shown for the first time, to be elevated
in patients with mechanical NP.
Chemokines and inflammatory cytokines may be used as biomarkers in
mechanical NP patients.
Elevated production of inflammation-associated chemokines and NO may
contribute to the genesis of NP.