Medical Conditions in the Reviewed Studies
Mazzone et al. (2001) examined inflammatory cytokines in a male cohort with exercise-induced myocardial ischemia (n = 78) and found no differences in proinflammatory cytokine levels (IL-1β, TNF-α, IL-6, and interferon-γ [IFN-γ]) between symptomatic and asymptomatic groups. However, the asymptomatic group had significantly higher levels of anti-inflammatory cytokines (IL-4 and IL-10).
The greatest number of studies comparing cytokines to the occurrence of pain was in individuals with FM, a chronic pain disorder of unknown etiology (Wang et al., 2008). In a prospective study, Wang et al. compared serum cytokine levels and pain intensity in patients with FM (n = 20) and healthy controls (n = 80) before and after receiving biopsychosocial therapy. At baseline, serum IL-8 and TNF-α levels were significantly higher in patients with FM compared with controls, whereas IL-6 levels were similar between groups. IL-8 levels remained higher in the patients with FM through follow-up, but TNF-α was reduced after 10 days and remained reduced through 180 days. IL-8 and TNF-α declined significantly from baseline through day 10 for both groups. In the FM group, it remained significantly lower through 180 days and, thus, may be related to analgesia. IL-8 correlated with pain intensity at 180 days but not at baseline, suggesting no direct correlation between cytokines and pain intensity.
Bazzichi and colleagues (2007) examined cytokine levels in women with FM (n = 80) and age-matched controls (n = 45). To control for the possible influence of psychological conditions known to be associated with increased cytokine levels, patients with FM were divided into three groups based on psychological status: (a) presence of depression, (b) anxiety disorder, or (c) no psychiatric illness. IL-1 was significantly lower in all three FM subgroups compared with controls. All FM groups, regardless of psychological status, had pain and higher levels of IL-8, IL-10, and TNF-α compared with the control group, suggesting a relationship between pain and cytokine levels independent of psychological status.
Iannuccelli et al. (2010) compared cytokines in women with FM (n = 51), women with tension-type headaches (n = 25), and healthy controls (n = 15). Serum IL-1ra, IL-6, IL-10, and TNF-α serum levels were higher in patients with FM compared with those with tension-type headaches and controls. There was also a significant correlation between IL-10 and Fibromyalgia Impact Questionnaire scores in the FM group, suggesting that a higher burden of symptoms results in the expression of anti-inflammatory cytokines.
Chronic Fatigue Syndrome
White et al. (2010) examined cytokine levels and the occurrence of pain in individuals with CFS. Individuals with CFS become fatigued and often experience body-wide pain after exercise, referred to as symptom flare (SF). Patients with CFS (n = 19) and healthy controls (n = 17) were enrolled, and all completed a moderate exercise protocol to induce SF. Eleven of the patients with CFS were categorized as high SF, and eight were categorized as low SF. Pain (myalgia) was measured at each time point using a numeric rating scale that ranged from 0–100. At baseline, the only biomarker significantly different between healthy controls and both low and high SF groups was CD40 ligand (CD40L). CD40L is part of the TNF family and is a marker of platelet activation. At 8 hours, the high SF group had increased proinflammatory and anti-inflammatory cytokines levels, whereas the low SF and control groups had decreases or small increases in both proinflammatory and anti-inflammatory cytokines (with the exception of IL-8). Importantly, an increase in IL-6 in both CFS groups was strongly associated with pain.
In vitro cytokines released from isolated peripheral blood mononuclear cells and serum cytokines were examined in women with Sjögren syndrome, an autoimmune syndrome typically characterized by dry eyes and dry mouth (Eriksson et al., 2004). Serum and in vitro cytokine levels were compared between women with and without self-reported pain with Sjögren syndrome, and each of those groups was compared with women with rheumatoid arthritis and healthy controls. Peripheral blood mononuclear cell-stimulated cytokines (IL1β, IL-6, IL-10, TNF-α, and IFN-γ) were significantly lower in women with Sjögren syndrome and myalgia compared with controls. Serum IL-18 levels were significantly increased in both the Sjögren syndrome and rheumatoid arthritis groups compared with controls. Serum IL-8 was increased in those with rheumatoid arthritis but not Sjögren syndrome.
Üceyler and colleagues (2007) examined the differences in expression of cytokines in patients with neuropathies compared with healthy controls. Patients with painful neuropathy had two-fold higher IL-2 messenger ribonucleic acid (mRNA) levels and TNF-α mRNA, and IL-2 and TNF levels compared with patients with painless neuropathy and healthy controls. Serum mRNA levels of the anti-inflammatory cytokine IL-10 were two-fold higher in patients with painless neuropathy compared with those with pain and healthy controls. IL-4 was 20-fold higher in patients with painless neuropathy and 17-fold higher in patients with painful neuropathy compared with healthy controls.
In a subsequent study, Üceyler and colleagues (2010) examined proinflammatory cytokine expression in patients with small-fiber neuropathy (n = 24) compared with healthy controls (n = 72). Patients with neuropathies were furthered classified into three categories: (a) definite small-fiber neuropathy, (b) probable small-fiber neuropathy, or (c) possible small-fiber neuropathy. Patients with small-fiber neuropathy had a two-fold higher expression of circulating IL-2, IL-10, and TGF-β1 mRNA levels and significantly higher release of IL-6 and IL-8 compared with controls. Patients with length-dependent small-fiber neuropathy had significantly more impairment because of pain than patients with non-length-dependent, small-fiber neuropathy.
Manero and Alcazar (2010) examined correlations with IL-8 and the occurrence of pelvic pain in a cohort of women undergoing invasive treatment for ovarian endometriomas. Pain severity was assessed using a visual analog scale, and women were categorized as having or not having pelvic pain. Serum IL-8 levels did not vary by group after adjusting for gravidity, length of menses, infertility, and body mass index.
Experimental Inflammation and Noxious Stimulation
Angst and colleagues (2008) conducted two experiments in which sunburn was induced in healthy volunteers to evaluate the role of cytokines in an acute inflammatory pain condition. In Experiment 1, two sunburn lesions were induced on the thigh. Pain measures were completed, and microdialysate samples were compared with a noninflamed skin site. After analyses, noxious heat was applied to the sunburn. In the second phase of the study, a crossover, double-blind design was used to allocate participants to receive 400-mg ibuprofen, 800-mg ibuprofen, or placebo. Pain testing occurred 35 minutes after ingestion of the medication or placebo. Significant increases were found in IL-6, IL-8, IL-10, granulocyte-colony stimulating factor, and macrophage inflammatory protein 1 beta. Noxious heat increased IL-7 and IL-13. Tissue levels of IL-1β and IL-6 did not change after the 400-mg dose of ibuprofen but decreased significantly (44% ± 32% and 38% ± 13%) after an 800-mg dose.
In summary, there were several key findings in the relationship between proinflammatory and anti-inflammatory cytokines and pain in a variety of medical conditions associated with inflammation. In six of the studies reviewed, baseline elevations in cytokines were reported in populations with painful conditions compared with controls, although the magnitude of change by which cytokines were altered varied (Bazzichi et al., 2007; Eriksson et al., 2004; Iannuccelli et al., 2010; Mazzone et al., 2001; Wang et al., 2008; White et al., 2010; Figure 3). For example, for TNF-α, there was no difference in levels between those with FM and healthy controls in two studies (Iannuccelli et al., 2010; Wang et al., 2008), but in other studies, there was a large difference in levels between patients with FM and controls (Bazzichi et al., 2007) and between patients with Sjögren syndrome and controls (Eriksson et al., 2004). In half of the studies reviewed, higher levels of proinflammatory markers (CRP, TNF-α, IL-2, IL-6, IL-8, IL-10, and CD40L) were associated with greater pain intensity/severity (Bazzichi et al., 2007; Üceyler et al., 2010 , 2007; Wang et al., 2008; White et al., 2010). The proinflammatory cytokines IL-8 and IL-18 were associated with pain in CFS, FM, and Sjögren syndrome only (Eriksson et al., 2004; Iannuccelli et al., 2010; White et al., 2010). In only 20% of the studies, proinflammatory cytokines (TNF-α, IL-1β, and IL-8) decreased after treatment for pain (Angst et al., 2008; Wang et al., 2008). Levels of cytokines (except IL-10) did not correlate with pain in FM (Iannuccelli et al., 2010), myocardial ischemia (Mazzone et al., 2001), or pelvic pain (Manero & Alcazar, 2010). Only one study examined the potential confounding influence of psychological disturbances on cytokines, and there was no significant association (Bazzichi et al., 2007).
The hypothesis that certain conditions, such as those reviewed here, would stimulate the release of inflammatory biomarkers and correlate to pain was partially supported. In addition, in half of the studies, higher levels of inflammatory cytokines were associated with more severe pain. However, these results were not found in all studies. Three variations to the hypothesized model (Figure 1) were noted following this review, including
- not all participants experienced pain in the presence of inflammatory cytokines;
- pain was associated with variations in cytokines, specifically no change in inflammatory cytokines, an increase in proinflammatory cytokines, and an increase in anti-inflammatory cytokines (depending on the study); and
- an increase in proinflammatory cytokines was related to the severity of pain.
Therefore, the hypothesized model of variables of interest in Figure 1 was revised and appears in Figure 4.
Findings showed that certain cytokines may be emerging or putative biomarkers of pain in specific chronic conditions. There was variability in the type of cytokine altered, as well as the magnitude of change, which may be attributable to differences in cytokine measurement modalities, timing of measurement, and type and duration of medical condition, which could markedly influence cytokine levels. The notion of cytokine biomarkers as a proxy measure of pain in conditions in which there is an absence or attenuation of the sensation of pain requires more investigation. In fact, only one study was found that included the measurement of cytokines in patients with both symptomatic and silent myocardial ischemia, but proinflammatory cytokine production did not differ between groups (Mazzone et al., 2001). However, anti-inflammatory cytokines (IL-4 and IL-10) were greater in patients experiencing pain, a potential relationship that requires more exploration. Because pain is a key symptom for many pathological conditions, but some patients do not have pain as a symptom, inflammatory biomarkers may hold potential in diagnosis and management of the condition.
It is important to note that cytokines present in different bodily fluids can be detected using classic solid-phase sandwich immunoassays, such as enzyme-linked immunosorbent assay (ELISA) or multiplex bead-based immunoassays (de Jager & Rijkers, 2006). Most of the studies reviewed used standard ELISA, whereas three studies used high-sensitivity, multiplex bead-based assays (Angst et al., 2008; Manero & Alcazar, 2010; Wang et al., 2008). The latter allows for simultaneous measurement of multiple cytokines. Both ELISA and bead-based assays capture or sandwich the cytokine of interest using two different antibodies (usually designated as a capture and reporter antibody). Both techniques are considered reliable and state-of the-art.
Strengths of the Studies
In some studies, rigorous control of possible confounders, such as anti-inflammatory medications, corticosteroids, smoking, inflammatory/autoimmune diseases, or infection, was built into the design (Angst et al., 2008; Mazzone et al., 2001). In some cases, small samples yielded statistically significant findings, suggesting large effect sizes (Angst et al., 2008; Eriksson et al., 2004; White et al., 2010). A strength of some studies was an a priori hypothesis and, in particular, strong rationale for the measurement of certain cytokines. For example, Angst et al. measured IL-1β, IL-6, and TNF-α and predicted that there would be significant increases with an inflammatory stimulus because other investigators had shown that these cytokines were involved in early proinflammatory responses and hyperalgesic action.
The studies included in this analysis were geographically diverse (e.g., United States and Europe), and several studies were longitudinal, providing an opportunity to analyze biomarkers over time and in response to treatment (Bazzichi et al., 2007; Wang et al., 2008; White et al., 2010). For instance, Wang et al. measured cytokines at baseline, 10 days, 21 days, and 180 days. This permitted an examination of what the authors labeled the “kinetic” course of cytokines over 6 months of treatment for the pain of FM.
Most studies did include a control group (Angst et al., 2008; Bazzichi et al., 2007; Eriksson et al., 2004; Iannuccelli et al., 2010; Üceyler et al., 2010 , 2007; Wang et al., 2008; White et al., 2010). Factors such as pH and presence of other antibodies can affect cytokine results; yet, the most common reason for discrepancy or variation among results can relate to short half-life and wide ranges in cytokine levels (Bienvenu, Monneret, Fabien, & Revillard, 2005). Hence, a healthy control group may reveal differences in cytokine levels that would otherwise go undetected.
Limitations of the Studies
Similar to the studies reviewed by Üceyler et al. (2011) in their systematic review of cytokines in FM syndrome and Parkitny et al. (2013) in their review and meta-analysis of inflammation in complex regional pain syndrome, several of the studies described here did not meet basic quality criteria. Limitations included small sample sizes (Angst et al., 2008; Eriksson et al., 2004; Üceyler et al., 2010; White et al., 2010), lack of gender balance (Angst et al., 2008; Mazzone et al., 2001), cross-sectional measures (Bazzichi et al., 2007; Eriksson et al., 2004; Iannuccelli et al., 2010; Mazzone et al., 2001), insufficient numbers of older adults (Angst et al., 2008; White et al., 2010), and a preponderance of Caucasian participants. In addition, factors with the potential to affect inflammatory processes and the release of inflammatory biomarkers (such as alcohol, smoking, body mass index, physical activity, medications, and inflammatory or autoimmune diseases) were often not controlled for through design or statistical analyses (Eriksson et al., 2004; Iannuccelli et al., 2010; Manero & Alcazar, 2010; Mazzone et al., 2001). It is possible that the significant association between cytokines and pain would disappear after controlling for obesity. It is well established that obesity is associated with increased inflammation (Panagiotakos, Pitsavos, Yannakoulia, Chrysohoou, & Stefanadis, 2005). In several studies, levels of cytokines were low or negligible (Iannuccelli et al., 2010; Üceyler et al., 2010, 2007); so, whether statistical significance translates to clinical significance remains unknown. In several studies, levels of numerous cytokines were not normally distributed, necessitating log transformations and the use of nonparametric testing (Iannuccelli et al., 2010; Üceyler et al., 2010, 2007).
Some studies used plasma versus serum as the source for cytokines, whereas others did not indicate whether samples were prepared as plasma or serum. Plasma, rather than serum, is preferred for cytokine measurement because serum concentrations are lower for TNF-α, IL-6, and IL-10, as well as for other cytokines, because of degradation during the clotting process (Wong et al., 2008). In addition, it is important to note that venous samples were obtained from venipuncture rather than an indwelling catheter because others have shown that cytokine production increases in response to an indwelling venous catheter—possibly because of local tissue injury at the site of catheter insertion (Gudmundsson et al., 1997). It is vital to state the methodology used for cytokine measurement. As noted above, most studies used a standard ELISA or multiplex bead-based immunoassays. Finally, studies with limitations were included because so few high-quality studies were available and also to allow readers to evaluate the validity of findings. In addition, it was shown just how disparate the studies were that were identified and how difficult it is to control for clinical and individual variables when conducting research on pain and biomarkers. These data may aid investigators in increasing the rigor of future studies, thus improving internal validity.
Results indicated that cytokines were altered in the presence of pain, but there was inconclusive evidence and an overall lack of data to support the use of cytokines as a biomarker of pain. The occurrence and severity of pain was associated with changes in both proinflammatory and anti-inflammatory biomarkers. The association between inflammatory markers varied in the direction and magnitude of expression, which in part may be explained by differences among studies in design, assays, disease condition and duration, acute versus chronic pain, variation in and severity of symptoms, and timing of measurement. We suggest that evidenced-based guidelines for measuring, analyzing, and reporting inflammatory cytokines be compiled to facilitate meta-analysis. To elucidate the potential impact of cytokines on the clinical diagnosis or conditions in which pain is a known symptom but may be absent, future nursing research should investigate the association between pain and biomarkers of inflammation and leukocyte migration and activation. Information on inflammatory status will enable nurses to better understand the features of painful conditions, provide information to patients, and support patients in decision making.
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Keywords:© 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
biological markers; cytokines; inflammation; pain