increased serum levels of interleukin 8 in (1).pdf

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Original Article

Increased serum levels of interleukin-8in patients with tension-type headache

Renan B Domingues1,2, Halina Duarte1, Natalia P Rocha3 andAntonio L Teixeira1,3

Abstract

Background and objectives: The pathophysiology of tension-type headache is not well understood. Increased peripheral

levels of pro-inflammatory cytokines may act as mediators of several chronic pain disorders. The aim of the present study

was to investigate the peripheral levels of chemokines in patients with tension-type headache.

Methods: This was a cross sectional study evaluating serum levels of chemokines in age and sex-matched tension-type

headache patients, ictally and interictally, and control participants. Beck Depression and Anxiety Inventories were

recorded. Serum levels of monocyte chemoattractant protein-1, macrophage inflammatory protein 1a, regulated onactivation, normal T cell expressed and secreted, eotaxin, eotaxin-2, interleukin-8, interferon gamma induced protein-10

were measured by enzyme-linked immunosorbent assay.

Results: A total of 96 participants (48 tension-type headache, 48 controls) were included. Interleukin-8 levels were

significantly increased in patients with tension-type headache when compared to controls (413.8 (123.41756.3) and

329 (107.8955.6), respectively, P 0.025). Anxiety and depression scores were higher in patients with tension-typeheadache but interleukin-8 increase in tension-type headache patients persisted after controlling for anxiety and depres-

sion symptoms. Patients with headache at the time of assessment had increased monocyte chemoattractant protein-1

levels when compared with patients without headache (2809.3 (11016122.2) and 1630.2 (669.331056.8), respectively

P 0.026). Patients with episodic and chronic tension-type headache had no significant differences in serum chemokineslevels.

Conclusion: Interleukin-8 was increased in tension-type headache and monocyte chemoattractant protein-1 was higher in

tension-type headache patients with headache, suggesting that pro-inflammatory mechanisms may participate in tension-

type headache pathophysiology.

Keywords

Tension-type headache, inflammation, chemokines, chronic tension-type headache, episodic tension-type headache,

depression, anxiety

Date received: 7 September 2014; revised: 16 October 2014; accepted: 20 October 2014

Introduction

Tension-type headache (TTH) is the most frequentform of headache; however, little is known about itsphysiopathology (1). Both peripheral and central mech-anisms have been implicated. Increased myofascial painsensitivity occurs in patients with TTH and peripheralsensitization of myofascial nociceptors may have a rolein this process. Sensitization of second-order neurons atdorsal horn/trigeminal nucleus and supraspinal neu-rons at thalamus and somatosensory cortex have alsobeen proposed to explain central increased pain sensi-tivity in TTH (2,3). Inammation may be implicatedin generating and maintaining pain in peripheral struc-tures and can also promote sensitization of central ner-vous system structures involved in nociception (4).

Therefore, it is reasonable to hypothesize that inam-mation may play a role in peripheral and central sensi-tization in patients with TTH.

1Neurosciences Postgraduation Program, Federal University of Minas

Gerais (UFMG), Brazil2CHRU, Pole Neurologie, Universite Lille 2, France3Interdisciplinar Laboratory of Medical Investigation, School of Medicine,

Federal University of Minas Gerais (UFMG), Brazil

Corresponding author:

Renan B Domingues, Service de Maladies et Pathologie Neurovasculaire,

CHRU Hopital Roger Salengro, 5e`me Etage, 59037 Rue Emilie Laine,

Lille 29055131, France.

Email: [email protected]

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DOI: 10.1177/0333102414559734

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Previous studies have investigated the peripherallevels of inammatory mediators in patients withTTH. Increased levels of interleukin-6 (IL-6) werefound in patients with both episodic and chronic TTH(10), being higher in chronic than in episodic TTH.Interleukin-1 beta and interleukin-18 levels wereshown to be increased in chronic TTH (11). These datasuggest that inammation participates in TTH patho-genesis. TTH has been associated with psychiatriccomorbidities, including depression (5) and anxiety dis-orders (6). Such disorders have been previouslyassociated with increased expression of pro-inammatory mediators as well, including cytokinesand chemokines (79). No study to date has evaluatedwhether the increase of pro-inammatory cytokines inTTH could be secondary to psychiatric comorbidities.

Chemokines are a family of small (810 kDa) proteinsthat can induce chemotaxis of inammatory cells to sitesof inammation and their activation. The family is com-posed of about 50 related molecules. According to thenumber and spacing of cysteine residues, four chemo-kines subfamilies have been identied: CC; CXC;C; CX3C. CC chemokines, the largest group, are char-acterized by the presence of two adjacent cysteine resi-dues near the N-terminus and CXC is the second largestgroup and presents a single amino acid between the rsttwo cysteine residues (12). No previous study has eval-uated chemokines in patients with TTH.

The aims of the current study were two-fold: (i) tocompare the serum chemokine levels between TTHpatients and healthy controls; (ii) to investigate whetherchemokine levels in TTH could be inuenced by psy-chiatric comorbidities such as depression and anxiety.

Methods

Patients and procedures

This was a cross-sectional study including patients withTTH and healthy controls. From June to September,2011, 48 patients with TTH, enrolled from the headacheclinic of the Santa Casa de Misericordia Hospital,Vitoria, Brazil, were recruited during their rst visit tothe clinic. A headache expert neurologist evaluated allpatients and the diagnoses were performed in accord-ance with the International Classication on HeadacheDisorders 2nd edition (13). All the patients were askedabout the presence of headache pain at the time of theevaluation and they were included irrespective of theirpain status (i.e. ictal or interictal pain state). Controlsubjects were recruited in waiting rooms at the sameoutpatient clinic by an active search carried out amongaccompanying subjects. Forty-eight age- and gender-matched healthy subjects with no history of previousprimary headaches were included in the control group.

Theminimal age for the inclusion of patients and controlsubjects was 18 years. Subjects with inammatory, infec-tious, allergic, autoimmune, hepatic, neurodegenerativeand neoplasic diseases were not included in the study, aswell as pregnant women and people in use of corticoster-oids or immunosuppressive drugs. This study receivedfull approval by the Ethics Committee on Research ofthe Escola Superior de Ciencias da Santa Casa deVitoria, Vitoria, Brazil and informed consent wasobtained from each participant.

Demographic (age, body mass index (BMI), race andmarital status) and headache features (time of disease,frequency of attacks in the last month) data were rec-orded. Anxiety was evaluated with the Beck AnxietyInventory (BAI) (14) and depression with the BeckDepression Inventory (BDI) (15). The anxiety symp-toms were classied as mild (BAI 815), moderate(BAI 1625) and severe (BAI> 25) (14). The depressivesymptoms were classied as minimal (BDI 09), mild(BDI 1018), moderate (BDI 1929) and severe (BDI3063) (15).

Chemokines assessment

Eight millilitres of whole blood was collected from allthe participants on the same day of the clinical assess-ment. In order to rule out any confounding factorcaused by circadian rhythm, all samples were collectedat the same time of the day, between 09:00 and 11:00hours. Serum was obtained after centrifugation andkept at 80C until analysis. For analysis, sampleswere thawed and excess of proteins removed by acid/salt precipitation, as routinely performed in our labora-tory (16). The concentration of chemokines was mea-sured using sandwich enzyme-linked immunosorbentassay to determine the levels of the chemokines mono-cyte chemoattractant protein-1(MCP-1), macrophageinammatory protein-1a (MIP-1a), regulated on acti-vation, normal T cell expressed and secreted(RANTES), eotaxin, eotaxin-2, interleukin-8 (IL-8),and interferon gamma induced protein 10 (IP-10)(DuoSet, R&D Systems, Minneapolis, MN, USA)(Table 1). Briey the capture antibody (concentrationprovided by the manufacturer) was diluted in phos-phate-buered saline (PBS), added to each well andleft overnight at 4C. The plate was washed fourtimes in PBS with 0.05% Tween 20. The plate wasblocked with 1% bovine serum albumin/PBS and incu-bated for 1 h at room temperature before washing fourtimes as described. The standards and samples wereadded and the plate incubated overnight at 4C. Onthe following day the detection antibody (concentrationprovided by the manufacturer) diluted in PBS wasadded, incubated for 2 h at room temperature and theplate washed. The colour reagent (o-phenylenediamine)

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was added to each well and the reaction was allowed todevelop in the dark for 15min. The reaction wasstopped with the addition of 1M H2SO4 to each well.The absorbance was read on a plate reader at 492 nm(Emax, Molecular Devices, MN, USA). The detectionlimit of the assays was 10 pg/mL. No cross reactivity orinterference was identied among the chemokinesassayed. The assay conditions were controlled, standar-dized and pre-optimized to ensure repeatability andreproducibility of the assays. All samples were assayedin duplicate and analyses were blinded. The inter-assaycoecient of variability (CV) and the intra-assay CVwere below 5% and 10%, respectively.

Statistical analysis

The data were analysed with SPSS, version 15.0 forWindows. The condence interval was 95% and thesignicance level was set at P< 0.05. Verication ofnormal distribution of data was performed using theKolmogorovSmirnov test. MannWhitney was usedfor the medians comparisons of continuous data.Demographic categorical characteristics were com-pared using chi-square analyses. Binary logistic regres-sion analysis was performed with the presence of TTHand the type of TTH as the dependent variables.

Results

Age, gender, ethnic distribution, marital status andBMI were not signicantly dierent between TTHpatients and controls (Table 2).

Patients with TTH had higher median BDI(P 0.041) and median BAI (P 0.014) whencompared with controls. Four (8.3%) TTH patientshad moderate to severe and 44 (91.7%) had no or mildanxiety symptoms. Forty-three TTH patients had min-imal to mild (89.6%) and 5 (10.4%) had moderate tosevere depression symptoms according to BDI score.

All the assays were above the limit detection for eachchemokine except one control subject and one TTH

patient for RANTES measure. By univariate analysisa signicant dierence in IL-8 levels was found betweenTTH patients and controls (P 0.039) (Figure 1).Eotaxin, eotaxin-2, IP-10, MCP-1, MIP-1a and

Table 1. List of names and abbreviations of measured

chemokines.

MCP-1 CCL2/MCP-1 (monocyte chemoattractant

protein-1)

MIP-1a CCL3/MIP-1a (macrophage inflammatoryprotein 1a)

RANTES CCL5/RANTES (regulated on activation,

normal T cell expressed and secreted)

Eotaxin CCL11/Eotaxin

Eotaxin-2 CCL24/Eotaxin-2

IP-10 CXCL10/IP-10 (Interferon gamma

induced protein 10)

Table 2. Clinical and demographic data of subjects with tension

type headache (TTH) and controls.

Controls n 48 TTH n 48 PAge (years) 38 (1964) 43.5 (1976) 0.838

Gender

Female (%) 46 (93.9) 46 (93.9)

Male (%) 3 (6.1) 3 (6.1) 1.0

Marital status

Married (%) 24 (49) 23 (46.9)

Single (%) 25 (50) 26 (53.1) 0.84

Ethnic distribution

White (%) 25 (51) 22 (44.9) 0.418

Brown (%) 15 (30.6) 24 (49) 0.063

Black (%) 9 (18.4) 3 (6.1) 0.064

BMI 25.48

(17.7149.31)

25.60

(18.7545.72)

0.920

BAI 4 (019) 7.5 (022) 0.014

BDI 5 (031) 8 (054) 0.041

Time of disease

(years)

20.5 14.8

Age of onset (years) 20 11.4HIT-6 62.5 7.9Frequency of

attacks per

month

9.4 7.3

Allodynia score 4.4 3.8P: MannWhitney test, BMI: body mass index; BAI: Beck Anxiety

Inventory; BDI: Beck Depression Inventory; HIT-6: headache impact test.

TTH

contr

ols100

125.9158.5199.5251.2316.2398.1501.2

631794.31000

P=0.039

pg/m

L

IL8

Figure 1. Comparison of interleukin-8 (IL-8) levels between ten-

sion-type headache (TTH) patients (n 48) and controls (n 48).

Domingues et al. 3


RANTES levels were not signicantly dierent betweenTTH patients and controls (P 1, 1, 0.081, 0.153 and0.540, respectively) (Table 3). After controlling for anx-iety and depressive symptoms, IL-8 levels remained sig-nicantly higher in patients with TTH when comparedwith controls (P 0.025). There were no signicant dif-ferences in chemokines levels when TTH patients werecategorized according to the severity of anxiety anddepressive symptoms. We performed the same analysisafter excluding male subjects (three controls andthree patients) and the results remained the same.Female TTH patients had signicantly higher IL-8levels than female controls, even after adjusting fordepression and anxiety (P 0.021).

Forty-two (87.5%) out of 48 patients with TTH wereclassied as having episodic TTH and six (12.5%) ashaving chronic TTH. Patients with chronic TTHwere older (median age 62.5 years, ranging from 47 to74 years) than patients with episodic TTH (median age35.5 years, ranging from 19 to 76 years), but there wereno signicant dierences in ethnicity, gender and mari-tal status groups in patients with episodic and chronicTTH (P 0.359, 0.289, and 0.592, respectively). By uni-variate analysis the levels of eotaxin were higher inpatients with chronic TTH (1261.33 pg/mL, 923.112073.56 pg/mL) than patients with episodic TTH(716.83 pg/mL, 163.841260.02 pg/mL) (P 0.029).MCP-1 levels were also higher in chronic TTH(3917.38 pg/mL, 2540.996122.25 pg/mL) than in epi-sodic TTH (1706.18 pg/mL, 669.3531056.79 pg/mL)(P 0.029). After adjusted analysis with binary logisticregression with age as covariate, there were no signi-cant dierences in eotaxin and MCP-1 levels betweenpatients with episodic and chronic TTH (P 0.682 and0.349, respectively).

Patients with headache at the time of assessment hadsignicantly higher MCP-1 levels (2809.3 pg/mL,1101.056122.25 pg/mL) than TTH patients withoutpain (1630.22 pg/mL, 669.3531056.78 pg/mL)(P 0.026) (Figure 2). This dierence was no longer

observed after excluding male patients from the ana-lysis (P 0.137). No other chemokine (eotaxin,P 0.112; eotaxin-2, P 0.751; IL-8, P 0.895; IP-10,P 0.751; MIP-1a, P 0.751; RANTES, P 0.895) aswell as no other clinical parameter (including age, head-ache impact test (HIT)-6, allodynia, number of head-ache days per month, BAI and BDI) was associatedwith the presence of pain.

Discussion

To the best of our knowledge this is the rst study toshow elevated IL-8 serum levels in patients with TTH.IL-8 is a chemokine produced by macrophages andother cell types exposed to dierent inammatory sti-muli. IL-8 promotes the preferential accumulation ofneutrophils in the sites of inammation. The pro-

TTH w

ith pa

in

TTH w

ithou

t pain

631

1000

1584.9

2511.9

3981.1

6309.6

10000

P=0.026

pg/m

L

CCL2/MCP-1

Figure 2. Comparison of CCL2/monocyte chemoattractant

protein-1 (MCP-1) levels between tension-type headache (TTH)

patients with (n 14) and without (n 34) headache pain.

Table 3. Chemokines levels in patients with tension type-headache (TTH) and controls.

Controls (n 48) TTH (n 48) P P*MCP-1 (pg/mL) 1251.5 (372.719485.4) 1812.4 (669.331056.8) 0.153 0.331

MIP-1a (pg/mL) 212.5 (13.517749.6) 183.1 (13.515592.7) 0.413 0.981

RANTES (pg/mL) 19331.1 (037986.1) 20052.4 (040027.6) 0.540 0.056

Eotaxin (pg/mL) 814.5 (395.114195.7) 819.1 (163.812460.1) 1.000 0.396

Eotaxin-2 (pg/mL) 1987.9 (623.410308.9) 2340.7 (501.68360.5) 1.000 0.673

IL-8 (pg/mL) 329.0 (106.8954.6) 412.8 (123.41756.3) 0.039 0.025

IP-10 (pg/mL) 515.9 (159.41188.0) 267.4 (133.050370.6) 0.081 0.576

P: MannWhitney test; P*: binary logistic regression analysis with adjusted for depression and anxiety scores; MCP-1: monocyte chemoattractant

protein-1; MIP-1a: macrophage inflammatory protein 1a; RANTES: regulated on activation, normal T cell expressed and secreted; IL-8: interleukin-8;IP-10: interferon gamma induced protein 10.

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inammatory role of this chemokine has been shown inseveral pathological conditions (17). This nding is inline with a previous study that demonstrated elevatedserum IL-6 levels in patients with TTH (10). It hasbeen shown that inammation may enhance nociceptivesignalling by aecting peripheral nerve and neuronalexcitability (18). One possible explanation of the ndingof increased IL-8 in TTH outside a headache attackmay be related with the interaction between stressand inammatory response. Emotional conict andstress are commonly reported in association with TTH(3). Recurrent or chronic stress may alter the functioningof the hypothalamic-pituitary-adrenal axis, thus enhan-cing pro-inammatory responses. There is considerableevidence showing that increased production of inam-matory biomarkers occurs in stress response and thismay be identied even in the prodromal stage of neuro-psychiatric disorders (19). Therefore, the interictalincrease in IL-8 could be part of a stress response. Thishypothesis requires further conrmation.

Elevated eotaxin and MCP-1 levels were found inpatients with chronic TTH; however, this dierencewas not signicant after adjusted analysis. It is possiblethat the small number of patients with chronic TTHprecluded demonstrating signicant dierences.Increased pro-inammatory cytokines were previouslyshown in patients with chronic TTH (11) and in patientswith other types of chronic primary headaches, such aschronic migraine and new daily persistent headache (20).The up regulation of pro-inammatory cytokines maybe implicated in neuroplastic changes of nociceptivecircuits, thus contributing to the chronication of pri-mary headaches. However, the relationship betweenchemokines and TTH chronication remains to bedetermined.

MCP-1 was increased in patients with headache atthe time of clinical assessment. The association betweenpro-inammatory cytokines and headache attacks hasbeen explored (21). An increase of IL-6, interleukin-10and RANTES was previously shown in migraineattacks (22). Bo et al. found increased MCP-1 in cere-brospinal (CSF) uid samples of patients with TTHand migraine attacks (23). In the same study, an ictalincrease of CSF interleukin-1 receptor antagonist andtransforming growth factor-1b, both anti-inammatorymolecules, was shown. All these ndings indicate thatmild ictal immune/inammatory changes occur in

primary headache disorders. One possible hypothesisfor the ictal increase of pro-inammatory moleculesin TTH is that miofascial pain triggers the release ofinammatory mediators leading to the excitation ofperipheral aerent nerves. Nevertheless, this hypothesisis unlikely since normal levels of inammatory medi-ators were found in tender trapezius muscle in patientswith chronic TTH (24). An alternative explanation isthat peripheral stimuli could activate the spinoreticu-lothalamic tract that extends rostrally to the hypothal-amus, resulting in the release of multiple circulatingmolecules such as corticotropin-releasing hormone,vasopressin, beta-endorphin and adrenocorticotrophichormone, which stimulate cortisol production that mayactivate peripheral leukocytes and the release of cyto-kines (25). This hypothesis is in line with a previousstudy suggesting that cytokine increase during head-ache attacks may represent a mild response to pain (23).

Anxiety and depression symptoms were higher inpatients with TTH. There is growing evidence that anx-iety and depression can lead to increased production ofpro-inammatory cytokines (79). However, in the pre-sent study, TTH patients with moderate and severeanxiety and depression scores had no increase inchemokine levels as compared with patients with mildanxiety and depression scores. Moreover, anxiety anddepression symptoms did not inuence the increasedlevels of IL-8 that showed to be independently asso-ciated with TTH.

This study has limitations. This is a cross-sectionalstudy not allowing the establishment of any causalassumption. The small number of patients with CTTHdoes not allow denite conclusions regarding whetherchemokine plays a role in TTH chronication. Anxietyand depression were assessed through self-administeredinventories, not a formal psychiatric evaluation. Also,we did not evaluate smoking status of the participants.The obtained samples were not fasting; however, weminimized the inuence of circadian rhythm by collect-ing all samples at the same time of the day.

In conclusion, the present study showed a mildincrease of IL-8 serum levels in TTH and an increaseof MCP-1 associated with TTH headache pain, sug-gesting that inammation has a role in TTH patho-physiology. Future and larger studies are still neededto assess the role of inammation in TTHchronication.

Clinical implications

. IL-8 levels were increased in tension-type headache.

. MCP-1 levels were higher in patients with headache at the time of assessment.

. There were no dierences in chemokine levels between episodic and chronic tension type headache.

. Pro-inammatory mechanisms may participate in tension-type headache pathophysiology.

Domingues et al. 5


Funding

This work was funded by CNPq and Fapemig, Brazil.Dr Domingues is the recipient of a CAPES post-doctoral

scholarship.

Conflict of interest

None declared.

Acknowledgements

We gratefully acknowledge the contribution of Cassio

Lacerda and Tarnara Campostrini in data collection.

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