Cytokines in gingival crevicular fluid in elderly rheumatoid arthritis patients in a population‐based cross‐sectional study: RANTES was associated with periodontitis

Abstract Objective We studied cytokines in gingival crevicular fluid (GCF) in a cross‐sectional population‐based cohort of rheumatoid arthritis (RA) patients ≥61 years of age with and without a diagnosis of periodontitis. Background data Earlier studies on cytokines in GCF in RA patients have not given clear results. Methods In a population‐based cross‐sectional study of patients ≥61 years of age, 233 RA patients were identified. 132 (57%) dentate RA patients participated. All participants received rheumatological and dental examinations, and had a panoramic radiograph taken. GCF was sampled on each patient. Interleukins 1‐β (IL‐1β), IL‐4, IL‐6, IL‐10, IL‐17A, tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), and chemokines RANTES/CCL5, eotaxin and monocyte chemoattractant protein (MCP‐1) were analyzed in GCF. These cytokines were stratified for periodontitis, age, gender, body mass index (BMI), smoking, and anti‐cyclic citrullinated protein (anti‐CCP) status. Binary logistic regression analyses with periodontitis as outcome were performed adjusting for the above mentioned confounding factors including anti‐rheumatic medication, disease duration and the cytokine in question. Results Periodontitis was diagnosed in 80/132 (61%) of study participants. The 110 RA patients not participating were older, had a higher mean erythrocyte sedimentation rate (ESR), had a higher mean DAS28ESR (Disease Activity Score 28 using ESR) and were less often on biologic treatment. Only RANTES was associated with periodontitis (p = .049, OR 1.001, 95% CI 1.000–1.002) in the binary logistic regression analyses. Conclusion In this population‐based elderly RA cohort, neither pro‐inflammatory nor anti‐inflammatory cytokines in GCF were clearly associated with a diagnosis of periodontitis.


| INTRODUC TI ON
Rheumatoid arthritis (RA) and periodontitis are two chronic diseases that have been associated with elevated levels of circulating proinflammatory cytokines and with clinical evidence of destruction of soft tissue and bone. 1,2 The inflammatory response in bacterially induced gingivitis and periodontitis results in gingival crevicular fluid (GCF) as an inflammatory exudate that can be collected at the gingival margin or within the gingival crevice or pocket. Since the 1960s, collection of GCF samples by a non-invasive method has been used extensively to determine sites of active disease, assess disease progression and to monitor effect of periodontal therapy.
Thus, a large number of cytokines, chemokines, bone remodeling enzymes, inflammatory mediators and tissue destruction enzymes in GCF can be used to assess periodontitis as a complement to the clinical evaluation. [3][4][5] Several pro-inflammatory and anti-inflammatory cytokines interact and correlate with each other. The GCF cytokine levels can be influenced by the different GCF sampling techniques used (absorption, microcapillary, washing), analysing techniques and demographics, such as smoking, medications, and comorbidities. 6,7 Two recent literature reviews have summarized studies performed in GCF in RA patients on cytokines, chemokines, enzymes, and tissue breakdown products stratified for periodontitis. 8,9 There were inconclusive results as to levels of interleukin-1β (IL-1β) in periodontitis in RA as summarized from several cross-sectional studies, and the same levels of interleukin-4 (IL-4) and interleukin-10 (IL- 10) between RA patients, periodontitis, and periodontally healthy patients. 8,9 Matrix metalloproteinase-8 (MMP-8), MMP-9, and tumor necrosis factor alpha (TNFα) levels were higher in periodontitis and in RA and MMP-8 levels increased with the severity of periodontitis. 8,9 These reviews included very heterogeneous studies with mostly very small convenience samples of RA patients, where RA patients with systemic and comorbid conditions were excluded. Several reviews and meta-analyses of cytokines in GCF in otherwise healthy patients with periodontitis report that cytokines such as IL-1α, IL-1β, interleukin-6 (IL-6), GMCSF (granulocyte macrophage colony stimulating factor), interleukin-12p40 (IL-12p40), interleukin-17 (IL-17), TNFα, monocyte chemoattractant protein (MCP1/ CCL2), matrix metalloproteases, bone remodeling products, prostaglandins, and macroglobulins are increased in periodontitis. [4][5][6][10][11][12][13] Tomas et al showed that the best predictors for periodontitis included IL-1α, IL-1β, and IL-17A in GCF. 11 In a case-control study, we have previously shown in this same RA patient cohort that RA was associated with a diagnosis of periodontitis with an odds ratio (OR) of 2.5 and that body mass index (BMI), periodontitis and female sex were associated with RA. 14 We studied pro-inflammatory and anti-inflammatory cytokines and chemokines in GCF in RA patients who were 61 years of age or older in a systematically sampled cross-sectional population-based cohort, stratified for having or not having periodontitis as defined and described below. Our null hypothesis was that the expression of pro-inflammatory cytokines in GCF from RA patients did not differ between those who had, or did not have, a clinical diagnosis of periodontitis. We were interested to study the cytokine and chemokine concentrations in GCF in a typical elderly RA patient population with long-standing disease, comorbidities, and systemic conditions and prescribed anti-rheumatic medication.  Figure 1 shows the study flowchart of identified and included participants.

| MATERIAL AND ME THODS
All individuals with RA were examined at the outpatient rheumatology clinic by rheumatologists. The medical records were reviewed by a rheumatologist (author MKS). Data on RA disease activity and current anti-rheumatic medications at inclusion were identified at the rheumatologists' visit and the data were confirmed from the

| Dental examination
A dental hygienist performed the clinical dental examinations. The examination also included a panoramic radiograph. Radiographs were assessed by a periodontist (author RGP) masked to clinical dental and medical data.
The following dental examination procedures were performed: 1. Measurements of probing pocket depths at four surfaces of teeth, and dental implants present.
2. The extent of bleeding on probing (BOP) was recorded within 30 s following probing of probing pocket depths (PPD) and assessed at the same sites as described above.
3. Dental plaque scores were recorded as described above.

| Definition of gingivitis and periodontitis
Periodontitis was defined as the clinical presence of bleeding on probing at >20% of recorded tooth surfaces, presence of >2 nonadjacent sites with a PPD ≥5 mm, presence of bone loss at ≥2 sites with a distance between CEJ-to bone level of ≥5 mm, or if evidence of a furcation invasion at molar teeth was found either clinically (grade II), or clearly visible on panoramic radiographs, and bone loss ≥5 mm at ≥30%. This definition of periodontitis was based on and adjusted to the current AAP/EFP classification of periodontitis ≥stage 2 to the extent possible due to differences in available data.

| Sampling of gingival crevicular fluid
Samples of GCF were collected as follows. Contamination with saliva was prevented by the placement of a cotton roll in the mucosal fold at each test site and supra-gingival plaque was removed with sterile cotton pellets. One sterile Perio Paper (Oraflow Inc) was placed into

| Laboratory assays
The

| Ethical approval
The

| Statistics
Mann-Whitney test was used to study the association of cytokines and periodontitis, gender and anti-CCP and the disease activity variables and demographics between periodontally healthy and periodontitis patients. Chi-square was used for categorical variables.
Spearman's correlation coefficient was used to study the correla-

| RE SULTS
Data from 132 individuals with RA were analyzed, giving a catchment of 57%. The flowchart of identified and included participants is shown in Figure 1. The mean percent BOP was 21%. Table 2 shows the levels (pg/ml) of the proinflammatory and antiinflammatory cytokines and chemokines in the 132 RA patients. Table 3 shows the levels of the cytokines (pg/ml) stratified for periodontitis. We could not see any differences between the cy-

| Binary logistic regression analyses
The binary logistic regression analyses showed that only RANTES

| DISCUSS ION
The data from the present cross-sectional study of an elderly  Interleukin-10 .522 Interleukin-17A  [4][5][6]11 In conclusion, we found no clear association of pro-inflammatory or anti-inflammatory cytokines in gingival crevicular fluid in elderly RA patients with periodontitis.

ACK N OWLED G EM ENTS
We wish to thank registered nurses Pia Jansson and Anna Andersson for help with the recruitment of the patients and Ms. Ulrika Isaksson for data management. We appreciate the work by RDS Ingrid Jonasson for the dental examinations. We also wish to thank Mr.
Hans Krona for help with the analyses of the blood samples. This work was supported by the Eklund Foundation (to SR), Region Blekinge (to MKS), the Crafoord Foundation (to MKS) and the Swedish Rheumatism Association (to MKS).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
All authors contributed with substantial contributions to conception and design of, and acquisition of data and analysis and interpretation of data, drafted the article and revised it critically for important intellectual content and finally approved the version to be published.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.