We found increased levels of IL-12, MIP-1β and CRP in the serum of subjects with work related MSD in the neck/shoulder region. We believe these findings represent an ongoing inflammation in these individuals.
Methodological considerations
As there was a time delay between answering the initial postal questionnaire and blood sampling in some cases, all subjects were interviewed regarding their current health status on the day of the blood test, and only subjects who were affected or unaffected, at the time of answering the questionnaire and on the day of the blood test, were included in the study. All calculations were performed on the results obtained from the interview and examination on the day of the blood test. In this way we were able form clearly distinct groups of subjects with and without MSDs.
The serum used for analysing the cytokines was frozen immediately after sampling. Since the recruiting of suitable subjects was a lengthy process, some samples were stored in the freezer for up to three years. When analysing the samples, we therefore paid special attention to any signs of possible drift in concentrations in the older samples. However, we observed no such drift, and therefore believe that the storage time did not affect the quality of the samples or the results obtained. Blood tests for CRP, on the other hand, were analysed continuously at the laboratory of the Skåne University Hospital. This resulted in a relatively crude analysis for our purpose. In spite of this, we found a statistically significant elevation of CRP levels in the MSD group and a correlation between CRP and pain intensity.
The age distributions in the affected and unaffected groups were comparable, and we found no correlations between age and any of the cytokines analysed or CRP. Age has, however, been suggested to correlate with inflammatory status[
16], and we therefore adjusted for this variable. Doing so did not significantly change the results.
Increased BMI is known to be associated with elevated CRP levels[
17], and obesity has also been reported to affect cytokine levels[
18] and, therefore, only subjects with BMI in the range 18-30 were included in the study. We found no correlations between the cytokines and BMI but, as would be expected, we did find a correlation between BMI and CRP. The two groups were, however, very similar with regard to BMI, and adjusting for BMI in a statistical model did not affect the result.
CRP is a marker of any type of inflammatory process, and there may be many reasons for an increase in CRP levels in an individual. Individuals with colds or other infections were, however, asked to return for blood sampling and examination at a later date. Our exclusion criteria also contained several disorders associated with inflammation. Thus, we believe that the elevated CRP levels found in the MSD group did in fact reflect an inflammatory component of their MSD.
IL-12 is released from classically activated macrophages following activation signals via e.g. TNF-α and GM-CSF[
19]. IL-12 levels were generally low in this study, and only four subjects had levels above the LOD. However, all of these were MSD subjects and we therefore observed a statistically significant difference between groups with and without MSDs. This finding is interesting, but it is not possible to draw any conclusions with such a limited number of samples above the LOD.
Performing multiple analyses may increase the risk of discovering seemingly significant findings by chance. This is a limitation of the study, but the observed increases in CRP and MIP-1β are in line with previously published results, and can be explained from a pathophysiological perspective.
As expected, many of the cytokines analysed were found to be correlated with each other. This is consistent with the belief that inflammatory cytokines can stimulate the production of one another. For example, IL-1α and TNF-α have been shown to induce cytokine gene expression and production[
20].
Implications
We were not able to verify the results of previous studies, in which the pro-inflammatory cytokines IL-1 or TNF-α were found to be elevated in animals and patients suffering from MSD caused by repetitive stress[
1,
4,
6]. In these studies, relatively newly developed disorders were examined, whereas our subjects had a long history of overuse MSD. However, increases were observed in pro-inflammatory chemokines (small cytokines), as discussed below.
Since previous studies have shown an increase in macrophages in the tissues, as well as an increase in the macrophage inflammatory protein 3a in serum, in animals performing repetitive tasks[
5], we included cytokines associated with macrophages in our analyses. The results showed a statistically significant increase in MIP-1β, a cytokine released by neutrophils, which acts as a chemokine and activating factor for monocytes and macrophages[
21]. This finding is consistent with the findings of Barbe et al.[
4]. Interestingly, MIP-1β was also correlated with the reported intensity of pain in the neck and shoulder in our subjects, suggesting that the level of MIP-1β increases with the severity of the disorder. To the best of our knowledge, MIP-1β has not previously been evaluated in subjects with overuse MSD. MIP-1α was also correlated to pain intensity, and showed a tendency towards elevated serum levels in the MSD group. However, this elevation was not statistically significant.
CRP is a well-known and reliable biomarker of inflammation and tissue injury[
17], and it has also been found to be elevated in subjects with repetitive work tasks[
22] and MSD[
6]. We found increased CRP levels in a substantial proportion (48%) of the affected subjects, indicating an underlying systemic inflammatory process in these individuals. Like MIP-1α and MIP-1β, CRP was correlated to the severity of neck/shoulder symptoms, but interestingly, MIP-1α, MIP-1β and CRP were not correlated with each other. It is possible that these biomarkers reflect different stages of an inflammatory process. The MSD subjects had both a long history of pain and a continuing exposure to repetitive work tasks. It could, therefore, be expected that we would find different stages of the inflammatory process within this group, and perhaps even within the same individual.
It has been suggested that repetitive and force demanding tasks lead to tissue fatigue failure[
7,
8], initially with inflammatory processes, followed by fibrotic processes. CTGF has been suggested as a potential mediator of fibrotic events[
23,
24], and has been shown to increase in the later stages of work-related MSDs[
25,
26]. Elevated levels of MIPs and CRP, but no difference concerning CTGF, between the subjects with and without MSD in the present study, may thus indicate that the affected subjects were in an inflammatory phase.
Since no differences were seen in cytokine levels between the control subjects with and without repetitive work tasks, we believe that the observed increase in inflammatory mediators seen in the MSD group is due to the musculoskeletal disorder resulting in pain, and not to the repetitive work itself.