Evaluation of the Human Pulmonary Activation-Regulated Chemokine (CCL18/PARC) and Alkaline Phosphatase (ALP) Levels in Iraqi Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is one of the chronic inflammatory autoimmune diseases which occurs as a result of unknown reasons. This study was conducted at Baghdad Teaching Hospital/City of Medicine, where blood samples were taken from 60 Iraqi patients with RA (49 females and 11 males) and these patients were matched by age and sex with 20 healthy controls (16 females and 4 males). Patients with RA were diagnosed by a consultant rheumatologist according to ACR / EULAR criteria in 2010. In this study the patients were divided into four groups as follows; the first group consisted of 12 patients treated with methotrexate (MTX), the second group consisted of 10 patients treated with etanercept, the third group consisted of 18 patients treated with a combination of MTX, etanercept and prednisolone, the fourth group consisted of 20 patients treated with MTX and etanercept. Enzyme linked immunosorbent assay (ELISA) was used to detect CCL18/PARC antibodies, while a spectrophotometer (Humalyzer2000) was used for the measurement of alkaline phosphatase (ALP). Serum levels of CCL18 / PARC showed a significant increase in RA patients compared with healthy controls (p ≤ 0.001). The levels of CCL18/PARC showed a significant correlation with disease activity (CDAI), except in RA patients treated with etanercept. There was also no significant correlation between CCL18/PARC and erythrocyte sedimentation rate (ESR). The results showed a significant increase in serum levels of alkaline phosphatase (ALP) was recorded in RA patients (with treatment) as compared to healthy controls (p ≤ 0.001).


Introduction
Rheumatoid arthritis (RA) is one of the types of autoimmune diseases of chronic inflammation, where the immune system attacks the endothelial cells of the joints causing chronic inflammation of the joints. Although RA affects the joints mainly and causes pain and damage in bones and cartilage, it has other effects on the lungs, eyes, heart and skin [1]. The main cause of RA is not yet known and it is also believed that environmental, genetic and aging factors have a role in etiology [2]. In a previous study, a statistical analysis was conducted by sex, age, year, cause and geography of many diseases, including rheumatoid arthritis from 2005 to 2015. It was found that RA affected up to about 24.5 million people [3]. The prevalence of RA worldwide is between (0.5 -1%), it also affects people of age 30 -50 years as well as children 16 years and above, and RA affects women 3 times more than men. The symptoms of RA appear in some patients in varying periods of time that may be from days to weeks or months and could even take several years. The passage of all this time without diagnosis and treatment leads to permanent deformations, including tendon rupture and erosion of cartilage and bones. The symptoms of RA are accompanied by pain and swelling of the joints, loss of weight, appearance of red circles under the skin, inability to breathe, pain in the chest and dry eye [4]. Increased RA disease activity leads to the destruction of cartilage and bones in the joints, also different types of treatments are used to reduce both disease progression and symptoms only because the effectiveness of drugs is limited and it does not eliminate RA [5][6]. Chemokine (CK) is one of the types of cytokines with a low molecular weight (6-14 k Da) and it's known by other names including; Human CCL-18 (CC-chemokine ligand-18) Pulmonary and Activation-Regulated Chemokine (CCL18/ PARC), Macrophage Inflammatory Protein-4 (MIP-4), Alternative Macrophage Activationassociated CC Chemokine 1 (AMAC-1) and Dendritic Cell Derived Chemokine 1 (DCCK1) [7]. Chemokine has important roles in determining the response of white blood cells and the assemblage of inflammatory cells in the site of inflammation. In addition, the excretion of CCL18/PARC is performed through the innate immune system. Although CCL18/PARC exerts chemical functions in the innate immune system, these functions are mainly related to the induction of the adaptive immune system [8]. The gene of CCL18/PARC is most analogous to that of CCL3. CCL18/PARC is present within several macrophages (MIPs), where it is located on the chromosome 17 [9]. Alkaline phosphatase (ALP) is one of the enzymes found in blood plasma and has a high molecular weight (140kDa). The effectiveness of ALP is increased in alkaline pH (9 -10.5) [10]. Alkaline phosphatase is found in some tissues of the body, mainly in the bones and liver and in a few amounts in the kidney, placenta, and bowel [11]. Besides that, ALP contains a group of enzymes that include; the isoenzyme produced by the bone (isoenzyme ALP-1) and the isoenzyme produced by the liver (isoenzyme ALP-2). The function of these enzymes is to facilitate the transport of metabolic products through membranes and lipids. ALP has a low hydrolysis group, which provides it with the ability to decompose a large group of phosphate esters by forming phosphate and alcohol ions. In addition, it is worthy to note that levels of ALP in the blood serum are high as a result of inflammation that occurs in the joint, which leads to an acceleration in the rate of bone turnover [12][13].
The aim of this study is to evaluate the levels of CCL18 / PARC and ALP in Iraqi patients with RA (with treatment), as compared with healthy controls, and to assess the role of CCL10/PARC as a possible marker of disease activity.

Materials and Methods
This study was conducted at Baghdad Teaching Hospital/City of Medicine, where blood samples were taken from 60 Iraqi patients with RA (49 females and 11 males) and these patients were matched by age and sex with 20 healthy controls (16 females and 4 males). Patients with RA were diagnosed by a consultant rheumatologist according to ACR / EULAR criteria in 2010. Blood samples were collected from September 2018 to January 2019. The medical history was collected from every patient and included the age, sex, chronic disease, duration of smoking, duration of the disease, duration of treatment, type of treatment (methotrexate, etanercept, and prednisolone), and the measurement of Clinical Disease Activity Index (CDAI). The disease activity was measured using the following equation; CDAI= Swollen 28-Joint Count + Tender 28-Joint Count + Patient Global disease Activity + Evaluator's Global disease Activity.
After that, patients were divided into four groups as follows; the first group consisted of 12 patients treated with MTX, the second group consisted of 10 patients treated with etanercept, the third group consisted of 18 patients treated with a combination of MTX, Etanercept and prednisolone, the fourth group consisted of 20 patients treated with MTX and etanercept. Five milliliters of blood were withdrawn from patients with RA and healthy controls. The blood samples were placed in test tubes (Gel Tube) and centrifuged at 3000 rpm for 10 minutes. After that, the serum was placed in a small tube (Eppendorf tube) and stored at temperature of -40°C. The serum was used to determine CCL18 / PARC level using the Sandwich-ELISA principle (Elabscience). The remaining serum was used to measure ALP levels activity by spectrophotometer (Humalyzer2000). In this study, patients with migraine, epilepsy, and thyroid and heart diseases were excluded.

Statistical analysis
Data analysis was performed using SPSS statistical program (Version/11.5; SPSS Inc., Chicago, IL). Analysis of variance (ANOVA) was used to determine whether there are any statistically significant differences between the means. Data were presented as mean ± Stander Deviation. Pearson's correlation (r-coefficient) was used between CCL18/PARC, disease activity and ESR. A pvalue of ≤0.05 was considered statistically significant.

Results and Discussion Human Pulmonary Activation Regulated Chemokine CCL-18/ PARC levels
In this study, the results showed a significant increase in serum CCL18 / PARC levels in RA patients as compared with healthy controls (Table-1). The cause of the high level of CCL18/PARC in RA patients was due to the infiltration of inflammatory cells such as stem cells, T cells, B cells and connective cells into the joints, causing the white blood cells to move to the site of inflammation and release cytokines that attacks the cells and releases other destructive substances. Over time, inflammation increases on the synovial membrane and pain occurs in the joints, associated with cartilage damage. It's also known that the synovial fluid and synovial tissue in RA patients contain high concentrations of several chemokines [14]. The results of this study were agreed with the previous studies [15][16], where the previous study showed a significantly increased in the serum levels of CCL18/PARC in RA patients compared with osteoarthritis patients (OA), and healthy controls [15] and also the previous study found a significantly increased in the serum levels of CCL18/PARC into the synovial fluid of RA patients compared with healthy controls [16]. The results in Figure-1 show that the monotherpay with MTX for RA patients caused reduction in the level of CCL18/PARC compared to those of the combination therapy, although the reduction did not reach a statistically significant level. The mean values of serum CCL18/PARC levels using monotherpay with MTX was 8327.54 ± 548.1pg/mL, monotherpay with Etanercept was 8330.79 ± 1117.96pg/mL, combination therapy with MTX and etanercept was 8589.13 ± 650.57pg/m), and combination therapy with MTX. etanercept, and prednisolone was 8649.60 ± 784.59pg/mL).

Correlation between CCL18/PARC concentrations in RA patients (with treatment) and disease activity (CDAI)
Data in Table-2 show a significant positive correlation between CCL18/PARC in RA patients (with treatment) and CDAI, while the correlation between CCL18/PARC in etanercept -treated RA patients and CDAI was non-significant. The results of the study were agreed with the previous studies [17][18], where the previous studies showed a positive correlation between CCL18/PARC levels and disease activity.

Erythrocyte Sedimentation Rate (ESR)
In this study, the results showed a significant increase in serum levels of ESR in RA patients (with treatment) as compared with healthy controls (Table-3). Erythrocyte Sedimentation Rate is one of the methods used to measure the level of the inflammation in the patient and the extent of stability of red blood cells. This study is consistent with a previous study which showed a significant increase in ESR levels in RA patients compared to controls [19].  Tables-3 and 4 show a non-significant correlation between CCL18/PARC and ESR levels in RA patients. The results of this study agree with those of a previous study, which shows a nonsignificant correlation between CCL18/PARC and ESR levels [15]. In this study, the results showed a significant increase in ALP levels of RA patients (with treatment) as compared with the healthy controls (Table-5). Increased serum ALP in patients with RA may be due to the change in the effectiveness of enzymes as a result of the damages in the bones and liver, which causes the release of these enzymes with more effectiveness than that observed in the normal conditions .The present study was agreed with a previous study. Previous study indicates the presence of a significant increase in serum levels of ALP in RA patients compared to healthy controls.
The authors explained their results by an ALP increase due to changes in the small joints and wrist joints. The compensatory mechanisms were reported to have an important role in restoring the deformed bones and, therefore, the activity of ALP increases [20].