# Six Years Follow-up of the Levels of TNF-α and IL-6 in  Patients with Complex Regional Pain Syndrome Type 1

Authors: Feikje Wesseldijk; Frank J. P. M. Huygen; Claudia Heijmans-Antonissen; Sjoerd P. Niehof; Freek J. Zijlstra
Journal: Mediators of Inflammation
 (2008)

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## Abstract

In an earlier study, levels of the proinflammatory cytokines TNF-α and IL-6 are higher in blisters fluid from the complex regional pain syndrome type 1 (CRPS1) side obtained at 6 and 30 months (median) after the initial event. The aim of this follow-up study is to determine the involvement of these cytokines in long lasting CRPS1. Twelve CRPS1 patients, with median disease duration of 72 months, participated. The levels of TNF-α and IL-6 were measured in blister fluid; disease activity was reevaluated by measuring pain and differences in temperature, volume, and mobility between both extremities. Differences in levels of IL-6 and TNF-α and mobility between both sides were significantly decreased. Pain and differences in temperature and volume were not significantly altered. No correlation was found between the cytokines and the disease characteristics. These results indicate that IL-6 and TNF-α are only partially responsible for the signs and symptoms of CRPS1.


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## Body

## 1. Introduction

Complex regional pain syndrome type 1 (CRPS1) is a disease of an extremity that usually
occurs as a complication after surgery or trauma, although spontaneous
occurrence is also described [1].The pathophysiology of CRPS1 is still not totally clear. In general, three mechanisms are thought to be involved: afferent mechanisms (e.g., neurogenic
inflammation) [2–4], efferent mechanisms (e.g.,
autonomic disturbances) [5], and central nervous system
mechanisms (e.g., cerebral plasticity) [6].
Based on our review of the literature regarding the CRPS pathophysiology, we
hypothesized that following a trauma or surgery, the normal sterile
inflammatory response runs out of control, and is perhaps initiated by a
genetic and/or acquired immunologic disorder [7].
Neuroimmune activation of cells in the peripheral nervous system, which
is part of the afferent mechanism, apparently results in central sensitization
and exacerbation of pain [8]. Neuropeptides, cytokines,
and other mediators are released during the inflammation [9, 10] and cause the prominent
signs and symptoms, which resemble inflammation; these include increased skin
temperature, edema, pain, loss of function, and redness [7, 11].Levels of TNF-α and IL-6 were previously shown to be elevated in fluid of artificially
induced skin blisters from the CRPS1 side in the initial stage of the disease [9, 12, 13]. These
observations suggest local inflammation. We hypothesized that in most patients local inflammation
would only be present during the first year of the disease, since the clinical
signs and symptoms of CRPS1 are expected to diminish over time in most
patients. Therefore, we predicted that the
formation of proinflammatory mediators (such as IL-6 and TNF-α) should
decline during the course of the disease. In an earlier study, we showed that
although the levels of TNF-α and IL-6 declined in the intermediate stage
of the disease, they were still significantly elevated in the CRPS1 extremity [13].
In the present study, we examine whether this decline in cytokine levels
continues during the course of the disease and whether this decline is
correlated with a possible improvement in disease activity as measured by
registration of pain, and by the measurement of differences in temperature,
volume, and mobility between the CRPS1 and contralateral extremities.


## 2. Materials and Methods

The protocol was approved by the medical ethics committee of the Erasmus MC
Rotterdam (MEC no. 1989.780/2001/24). Guidelines according to the Declaration
of Helsinki (amended version of 2002) and Good Clinical Practice (ICH/GCP
version 1996) were followed. Data collection and calculations were performed
according to guidelines for registration of personal data.


### 2.1. Patients

Sixty-six patients with CRPS1 in one extremity for 7 months
after the initial event participated in several studies conducted between April
2001 and February 2004 (T0) to investigate the pathophysiology of CRPS1 or the
effects of specific treatments for CRPS1 [9, 12, 14]. At the time of the first follow-up study in
2004, 25 patients with CRPS1 with a median disease duration of 30 months after
the initial event agreed to participate (T1) [13]. In 2007, these 25 patients were
again asked to participate. One patient had died, one patient appeared to have
CRPS type 2, and 11 patients chose not to participate again. In the end, 12
patients with CRPS1 were included in this study (T2).For this study, we used the results obtained at baseline (T0), first
follow-up measurement (T1), and second follow-up measurement (T2) from only
these 12 patients. All 12 patients fulfilled the CRPS1 criteria by Bruehl et
al. [15] at the first measurement (T0), performed shortly after the
initial event (median 4 months) which resulted in the development of the
disease.


### 2.2. Pain Assessment

The intensity of pain was assessed by using a visual
analoge scale (VAS) recorded in 0–100 millimeters [16]. The McGill Pain Questionnaire,
Dutch Language version (MPQ-DLV), was measured by counting the total number of
words chosen from 20 items [17].


### 2.3. Temperature Measurement

Skin temperature was measured using an infrared tympanic probe thermometer, First
Temp Genius (Sherwood Medical, Crawley, Sussex, UK) [18]. Measurements were obtained
on the dorsal aspect of the hand in a matrix of five points. The difference in
mean temperature between the CRPS1 and contralateral extremities was
calculated, and the data were expressed with respect to the temperature of the
unaffected hand.


### 2.4. Assessment of Volume

Volume was measured with a volumeter, which measures the amount of water displaced by
immersion of a body part [19].
The difference between the CRPS1 and contralateral extremities was calculated
as a percentage of the contralateral extremity.


### 2.5. Assessment of Mobility

Mobility was assessed by measuring the active range of
motion (AROM), which is defined as the arc of motion requiring muscle power to
achieve the motion of a joint [20, 21]. The
AROM on the CRPS1 extremity was multiplied by 100 and divided by the AROM on
the contralateral extremity to derive the percentage of normal mobility.


### 2.6. Blisters

Artificial skin blisters were
induced using a suction method [9, 14, 22]. A skin
suction chamber was positioned on the skin of the CRPS1 and contralateral
extremities. A vacuum of 300 mm Hg was applied with an Atmoforte 350 A aspirator
pump (ATMOS Medizintechnik, Lenzkirch, Germany). After 15 minutes, the vacuum was reduced to 250 mm Hg,
and after another 15 minutes
it was reduced to 200 mm Hg. This negative pressure was maintained for 2–2.5 hours. The
blisters created were punctured, and fluid was pooled from each side into a 1.5 mL
Eppendorf conical polypropylene tube and centrifuged for 5 minutes at 1600×g. All samples were stored in 1 mL
conical polypropylene tubes at −80°C until analysis [9, 22].


### 2.7. Cytokine Assays

Blister fluid samples were diluted 4-fold
in appropriate calibrator dilutent assay buffer for the direct measurement of
cytokines. Cytokine assays were performed following the manufacturer's protocol,
PeliKine human ELISA kits for IL-6 [M1906] and TNF-α [M1920] (CLB, Amsterdam,
the Netherlands). The standard curve ranges and mean calculated zero signal ±3 (SD) were 0–80 and 0.3 pg/mL
for IL-6 and 0–1000 and 1 pg/mL
for TNF-α. The absorbance per well was measured at 450 nm with a Medgenix EASIA
reader. Sample concentrations were calculated using the appropriate standard
calibration lines and the Softmax software of the reader.


### 2.8. Statistical Analysis

Data were analyzed with SPSS for
Windows, version 14.0. To determine whether the 12 CRPS1 patients from this
study were a representative group of the initial 66 CRPS1 patients who started
the follow-up study, the one-way ANOVA test was used for a comparison between
groups for the proinflammatory cytokines, and the outcome parameters pain,
temperature, volume, and mobility. To determine if the differences in means
for these selected parameters were significantly different from 0, we applied
the one-samplet-test for comparisons of the
levels of the proinflammatory cytokines between
both extremities, and to the differences in the
outcome parameters of temperature, volume, and mobility. Mixed
model analysis was used to compare the differences between both extremities to
each other for the levels of the cytokines, VAS, McGill, temperature, volume,
and mobility between each time point. Correlations
between the differences in levels of IL-6 and TNF-α and the outcome parameters
were calculated using the Pearson correlation test. Significance
was accepted at the P<.05 (two-sided) level.


## 2.1. Patients

Sixty-six patients with CRPS1 in one extremity for 7 months
after the initial event participated in several studies conducted between April
2001 and February 2004 (T0) to investigate the pathophysiology of CRPS1 or the
effects of specific treatments for CRPS1 [9, 12, 14]. At the time of the first follow-up study in
2004, 25 patients with CRPS1 with a median disease duration of 30 months after
the initial event agreed to participate (T1) [13]. In 2007, these 25 patients were
again asked to participate. One patient had died, one patient appeared to have
CRPS type 2, and 11 patients chose not to participate again. In the end, 12
patients with CRPS1 were included in this study (T2).For this study, we used the results obtained at baseline (T0), first
follow-up measurement (T1), and second follow-up measurement (T2) from only
these 12 patients. All 12 patients fulfilled the CRPS1 criteria by Bruehl et
al. [15] at the first measurement (T0), performed shortly after the
initial event (median 4 months) which resulted in the development of the
disease.


## 2.2. Pain Assessment

The intensity of pain was assessed by using a visual
analoge scale (VAS) recorded in 0–100 millimeters [16]. The McGill Pain Questionnaire,
Dutch Language version (MPQ-DLV), was measured by counting the total number of
words chosen from 20 items [17].


## 2.3. Temperature Measurement

Skin temperature was measured using an infrared tympanic probe thermometer, First
Temp Genius (Sherwood Medical, Crawley, Sussex, UK) [18]. Measurements were obtained
on the dorsal aspect of the hand in a matrix of five points. The difference in
mean temperature between the CRPS1 and contralateral extremities was
calculated, and the data were expressed with respect to the temperature of the
unaffected hand.


## 2.4. Assessment of Volume

Volume was measured with a volumeter, which measures the amount of water displaced by
immersion of a body part [19].
The difference between the CRPS1 and contralateral extremities was calculated
as a percentage of the contralateral extremity.


## 2.5. Assessment of Mobility

Mobility was assessed by measuring the active range of
motion (AROM), which is defined as the arc of motion requiring muscle power to
achieve the motion of a joint [20, 21]. The
AROM on the CRPS1 extremity was multiplied by 100 and divided by the AROM on
the contralateral extremity to derive the percentage of normal mobility.


## 2.6. Blisters

Artificial skin blisters were
induced using a suction method [9, 14, 22]. A skin
suction chamber was positioned on the skin of the CRPS1 and contralateral
extremities. A vacuum of 300 mm Hg was applied with an Atmoforte 350 A aspirator
pump (ATMOS Medizintechnik, Lenzkirch, Germany). After 15 minutes, the vacuum was reduced to 250 mm Hg,
and after another 15 minutes
it was reduced to 200 mm Hg. This negative pressure was maintained for 2–2.5 hours. The
blisters created were punctured, and fluid was pooled from each side into a 1.5 mL
Eppendorf conical polypropylene tube and centrifuged for 5 minutes at 1600×g. All samples were stored in 1 mL
conical polypropylene tubes at −80°C until analysis [9, 22].


## 2.7. Cytokine Assays

Blister fluid samples were diluted 4-fold
in appropriate calibrator dilutent assay buffer for the direct measurement of
cytokines. Cytokine assays were performed following the manufacturer's protocol,
PeliKine human ELISA kits for IL-6 [M1906] and TNF-α [M1920] (CLB, Amsterdam,
the Netherlands). The standard curve ranges and mean calculated zero signal ±3 (SD) were 0–80 and 0.3 pg/mL
for IL-6 and 0–1000 and 1 pg/mL
for TNF-α. The absorbance per well was measured at 450 nm with a Medgenix EASIA
reader. Sample concentrations were calculated using the appropriate standard
calibration lines and the Softmax software of the reader.


## 2.8. Statistical Analysis

Data were analyzed with SPSS for
Windows, version 14.0. To determine whether the 12 CRPS1 patients from this
study were a representative group of the initial 66 CRPS1 patients who started
the follow-up study, the one-way ANOVA test was used for a comparison between
groups for the proinflammatory cytokines, and the outcome parameters pain,
temperature, volume, and mobility. To determine if the differences in means
for these selected parameters were significantly different from 0, we applied
the one-samplet-test for comparisons of the
levels of the proinflammatory cytokines between
both extremities, and to the differences in the
outcome parameters of temperature, volume, and mobility. Mixed
model analysis was used to compare the differences between both extremities to
each other for the levels of the cytokines, VAS, McGill, temperature, volume,
and mobility between each time point. Correlations
between the differences in levels of IL-6 and TNF-α and the outcome parameters
were calculated using the Pearson correlation test. Significance
was accepted at the P<.05 (two-sided) level.


## 3. Results

The group of 12 CRPS1 patients was a good representation of the original group of 66 CRPS1 patients
with which we initiated the follow-up study 6 years ago. The groups were equal
for difference in levels of IL-6 (P=.60) and TNF-α (P=.72). They were also equal for the McGill
pain score (P=.41),
temperature difference (P=.41),
volume difference (P=.48),
and mobility (P=.38).
The two groups were only significantly different from each other for VAS pain (P=.03). The characteristics
and medication use of the 12 patients with CRPS1 in one extremity for 6 years (median)
who were examined 3 times during the follow-up of their CRPS1 are presented in
Table 1.Table 1
Characteristics of
the study population.
Patient characteristicValueGender: male/female3/9Side: right/left6/6Cause: fracture/accident/surgery/spontaneous6/2/4/0T0T1T2Age in years52 (48–56)54 (51–58)57 (54–62)Duration of CRPS1 in months4 (3–14)35 (21–48)72 (59–86)Disease-related medication (number of patients)Nonsteroid anti-inflammatory drugs (NSAIDs)610Opiates422Antioxidants810Vasodilators100Muscle relaxants100Antidepressants110Benzodiazepines200Antiepileticum101Data are presented as then or median (interquartile range).The IL-6 and TNF-α levels in blister fluid at all time intervals are
presented in Table 2. The differences in levels of IL-6 between both
extremities did not significantly change at T1 compared to T0 (P=.531) and at T2
compared to T0 (P=.063).
However, the differences in levels did significantly decrease at T2 compared to
T1 (P=.028).
The differences in levels of TNF-α between both extremities did not
significantly change at T1 compared to T0 (P=.892) and at T2 compared to T0 (P=.153). The decrease
in differences from T1 to T2 almost reached significance (P=.064). At the T0 and T1 measurement, the difference in
levels of IL-6 and TNF-α between the CRPS1 side and the contralateral side in
blister fluid was significantly different from 0. However, at the T2
measurement, no significant differences between the two sides were evident
(Figures 1(a) and 1(b)) for the 12
CRPS1 patients.Table 2
Levels of IL-6 and TNF-α in blister fluid obtained from the CRPS1 and contralateral extremities
at first measurement (T0), second measurement (T1), and third measurement (T2). Data are presented as mean (range).
T0T1T2CRPS1ContralateralCRPS1ContralateralCRPS1ContralateralIL-6 (pg/mL)116* (5–662)8 (1–36)80* (1–346)2 (0–5)22 (4–78)20 (3–61)TNF-α (pg/mL)66* (1–359)31 (1–258)56* (3–176)16 (2–80)38 (9–81)47 (10–142)*Wilcoxon signed ranks
test P<.05 (CRPS1 versus contralateral).Differences in levels of IL-6 and in TNF-α in blister fluid between the CRPS1 and
contralateral extremities over the course of CRPS1. Blister fluid was collected
as described in materials and methods (see Section 2) and IL-6 (panel a) and TNF-α
(panel b) levels (pg/mL) in the CRPS1 and the contralateral limbs were measured by
ELISA. The data obtained from the same 12 patients at each time period are
expressed as the difference in IL-6 or TNF-α levels between the two sides (log pg/mL).  Each time point shows the mean ± the
standard deviation. The P values
represent the deviation from no difference (0).
(a)(b)Also the signs and symptoms of impairment were measured at
T0, T1, and T2 in terms of pain and differences in temperature, volume, and
mobility between the CRPS1 and the contralateral extremities. The VAS pain did
not significantly change during the course of the disease in these 12 patients
(P=.472). The McGill pain score improved significantly at T1
compared to T0 (P=.006),
but pain was significantly worse at T2 compared to T1 (P=.041). Furthermore,
no significant improvement was seen at T2 as compared to T0 (P=.567). The VAS pain
and the McGill pain score significantly differed from 0 during all 6 years of
follow-up (Figures 2(a) and 2(b)).The assessment of pain over the course of CRPS1 disease
as measured by the Visual Analogue Scale is shown in panel (a).
Panel (b) shows the assessment of pain as measured by the McGill
Pain Questionnaire. Panel (c), (d), and (e), respectively, show the
absolute differences in skin surface temperature, the absolute
differences in volume, and the differences in AROM between the
CRPS1 and contralateral sides in CRPS1 patients over the course of
disease. The data obtained from the same 12 patients at the 3
points of measurement were collected as described in materials and
methods and are expressed as the mean±
standard deviation. The P
values represent the deviation from no difference (0)
(a)(b)(c)(d)(e)The absolute difference in temperature and volume
between both sides did not change during the course of the disease (P=.204 and P=.509,
resp.). The difference between the CRPS1 and contralateral side did vary
significantly from 0 at all three moments of measurement for temperature and
volume (Figures 2(c) and 2(d)).
Mobility improved significantly at T1 and T2 as compared
to T0 (P=.002
for both), with no significant increase in AROM at T2 compared to T1 (P=.353). The
difference in mobility between both sides was significantly different from 0 at
T0 and T1, but no significant difference was found at T2 (Figure 2(e)).No correlation was found between the differences in levels of
the proinflammatory cytokines and the other disease-related parameters pain and
differences between both extremities for temperature, volume, and mobility
during the course of the disease.The total use of disease-related medication was divided in 8 categories
(see Table1). The total number of patients using medication as well as the variety in
types of medication decreased during the course of the disease.


## 4. Discussion

In a six years
follow-up study in 12 CRPS1 patients, we found a decrease in the extent of the
differences in levels of TNF-α and IL-6 in blister fluid. After disease
duration of 4 months (T0) and of 3 years (T1), the levels of these cytokines
were significantly higher in the CRPS1 extremity compared to the contralateral
extremity. However, after 6 years (T2) the differences in the cytokine levels
between the two extremities were not significantly different. In contrast, the difference
in mobility between both sides was significantly improved after 6 years. Pain
and the differences in temperature and volume were not significantly altered
during the course of the disease. No correlation was found between the
proinflammatory cytokines and the disease characteristics.Because
TNF-α and IL-6 are proinflammatory cytokines, one might expect that these
markers of inflammation would be directly related to the characteristics of
inflammation: pain, temperature increase, edema, and loss of function. In this study, the levels of TNF-α and IL-6 were
diminished during the course of the disease, but no clear improvement of
inflammatory signs was found. Most of the 12 patients still reported much pain. The
VAS score did not change during the course of the disease, and while the McGill
Pain score improved at first, it worsened again at the third measurement. The
absolute temperature difference tended to diminish during the course of the
disease; however, this was not significant. The volume difference also did not
change significantly during the course of the disease. Only the AROM improved
significantly after 6 years; the patients only described some stiffness of the
joints.No improvement of the signs and symptoms of
inflammation was found as described above, however after six years, a decline
in the number of patients using medication that could counteract inflammation
has been observed, such as NSAIDs, opiates, and antioxidants. We concluded that
the normalization of inflammatory mediators after six years was not affected through
pharmacological intervention, but due to a diminution of disease activity.An explanation for a decrease in differences in the levels of
TNF-α and IL-6 between both sides during the course of CRPS1 is a spreading of
the disease from the CRPS1 side to the contralateral extremity. This spreading
could also be explained by the fact that the levels in the CRPS1 side are
increased during the course of the disease. However, the increased levels of
the contralateral side measured 6 years after the initial event do not even
come close to the high levels of TNF-α and IL-6 measured in the CRPS1 side 4
months and 3 years after the initial event. Furthermore, the levels of IL-6 of
both sides at T2 are within the normal range of the levels measured in blister
fluid from healthy controls (mean 16 pg/mL) [23]. These findings do not support the theory of spreading.
Finally, one would expect that the differences between both sides in disease
activity would decrease during the course of the disease when there is
spreading. This is not confirmed in this follow-up study.In the present
study, in contrast with the diminished levels of the proinflammatory cytokines
during the course of the disease, signs of inflammation were found in all three
stages of the disease. In an earlier study, during the first two years of
CRPS1, about half of the patients showed no inflammatory signs [24], but did fulfil the criteria
of Bruehl et al. [15]. Several other studies confirmed that observation, even describing a primarily cold
CRPS1 [4, 10, 25, 26].An explanation for finding a temperature increase and decrease already at the
onset of CRPS1 is that blood flow; and thus changes in skin temperature are
the results of not only afferent mechanisms, such as local mediators, but also
efferent mechanisms. Disturbances in
central temperature regulation could result in altered (local) temperature of
the injured extremity [27–29]. Recently, we
found evidence that temperature changes also might be partly caused by the crosstalk
in the vascular system between higher levels of cytokines and the nitric
oxide/endothelin-1 (NO/ET-1) balance [30, 31]. Furthermore, blood flow and tissue-blood distribution could be
diminished, partly due to disuse of the extremity [13].A possible explanation for finding edema and atrophy
during all stages of the disease is that edema is not only affected
by TNF-α and IL-6, but also by other inflammatory mediators such as calcitonin
gene-related peptide (CGRP) and substance P (SP), and related mechanisms [32, 33].No correlation was found between the differences in levels of the cytokines and pain and
differences between both extremities for temperature, volume, and AROM for all
three times of measurement. In an earlier study, we found similar results [24]. Perhaps IL-6 and TNF-α are
only partially responsible for the permanent damage in CRPS1, expressed as
pain, and changes in temperature, volume, and mobility. Other mediators or a
combination of mediators, such as nitric oxide [30] and/or amino acids [34, 35] or other mechanisms [7], may also play a role in the
pathophysiology of CRPS1 and may explain in part the course of the disease.



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