Yonsei Med J.  2015 Jul;56(4):1134-1142. 10.3349/ymj.2015.56.4.1134.

Chemokine Signaling Pathway Involved in CCL2 Expression in Patients with Rheumatoid Arthritis

• Zhang L
• Yu M
• Deng J
• Lv X
• Liu J
• Xiao Y
• Yang W
• Zhang Y
• Li C

• KMID: 2366358
• DOI: http://doi.org/10.3349/ymj.2015.56.4.1134

Abstract

PURPOSE
Rheumatoid arthritis (RA) is an inflammatory joint disorder, the progression of which leads to the destruction of cartilage and bone. Chemokines are involved in RA pathogenesis. In this study, we investigated the chemokine signaling pathway associated with CCL2 in peripheral blood (PB) and synovial tissues (ST) of RA patients based on our previous work about chemokine signaling pathway involved in the activation of CCL2 production in collagen-induced arthritis rat ST.
MATERIALS AND METHODS
Total RNA was isolated from PB leukocytes and synovium of the knee joint in both RA patients and control populations. Real-time polymerase chain reaction was used to determine CCL4, CCR5, c-Jun, c-Fos, and CCL2 expressions. Serum level of CCL2 was assessed by enzyme-linked immunosorbent assay, and the production of CCL2 in ST was analyzed immunohistochemically.
RESULTS
The expressions of CCL4, CCR5, c-Jun, c-Fos, and CCL2 messenger RNA in RA patients were significantly higher than those in healthy controls, both in ST and on PB leukocyte. Serum CCL2 levels were elevated in RA patients. Histological examination of rheumatoid joints revealed extensive CCL2 expression in RA ST.
CONCLUSION
CCL2, CCL4, c-Jun, c-Fos, and CCR5 may play an important role in the recruitment of PB leukocytes into the RA joints. These data provide evidence that the chemokine signaling pathway is involved in CCL2 expression in RA patient tissues, which may contribute to chronic inflammation associated with RA. Targeting this signaling pathway may provide a novel therapeutic avenue in RA.

Keyword

CCL2; leukocytes; rheumatoid; arthritis; synovial membrane

MeSH Terms

Adult
Animals
Arthritis, Rheumatoid/*blood/metabolism
Case-Control Studies
Chemokine CCL2/*blood/metabolism
Chemokines/metabolism
Enzyme-Linked Immunosorbent Assay
Female
Gene Expression
Humans
Male
Middle Aged
RNA, Messenger/genetics/metabolism
Rats
Real-Time Polymerase Chain Reaction
Signal Transduction
Synovial Membrane/*metabolism
Chemokine CCL2
Chemokines
RNA, Messenger

Figure

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  Fig. 1 Real-time polymerase chain reaction analysis of CCL2, CCL4, CCR5, c-Fos, and c-Jun mRNA levels on peripheral blood leukocytes. The relative mRNA expression levels were normalized to the levels of GAPDH. The significance of differences was determined using Student's t-test. *p<0.05, n=10 (open bars healthy controls, coarse stippling bars rheumatoid arthritis patients). mRNA, messenger RNA.

• 

  Fig. 2 Real-time polymerase chain reaction analysis of CCL2, CCL4, CCR5, c-Fos, and c-Jun mRNA levels in synovial tissue. The relative mRNA expression levels were normalized to the levels of GAPDH. The significance of differences was determined using Student's t-test. *p<0.05, n=6 (open bars healthy controls, coarse stippling bars rheumatoid arthritis patients). mRNA, messenger RNA.

• 

  Fig. 3 Concentrations of CCL2 protein in serum, assessed by enzyme-linked immunosorbent assays technique. The significance of differences was determined using Student's t-test. *p<0.05, n=11 (open bars healthy controls, coarse stippling bars rheumatoid arthritis patients).

• 

  Fig. 4 Immunohistochemical study of CCL2 expression in synovial tissue of knee joints. Photomicrographs were taken from healthy controls (A-D), rheumatoid arthritis patients (E-H). (A, C, E, and G) CCL2 immuoreactivity (brown staining). (B and F) Background staining with CCL2 antibody omitted (blue staining). (H) Maximal inflammatory cell infiltration (hematoxylin and eosin staining in D and H; avidin-biotin-peroxidase staining in A-G. Original magnification ×400 in A, B, D, E, F, and H; ×1000 in C and G). Bar=40 µm.

• 

  Fig. 5 The gene expressions that we have confirmed are shown in black, and confirmed pathways are shown with a solid line. The deductive results that may be involved are shown in coarse stippling. mRNA, messenger RNA; AP-1, activator protein.

• 

  Fig. 6 (A) Schematic representation of CCL4/CCR5/c-Jun and c-Fos/CCL2 signaling pathway in RA. Red color indicates gene increased expression, the darker the higher expression. (B) Signal transduction cascades in rheumatoid arthritis and factors that stimulate the CCL2 transcription. Signaling molecules that we have confirmed are shown in coarse stippling, and confirmed pathways are shown with bold arrows. Suspected factors that may be involved are shown in white, and potential pathways are shown with dashed arrows. DAG, diacyl glycerol; PIP2, phosphatidylinositol 4,5-trisphosphate; PKC, protein kinase C; PYK2, prolinerich tyrosine kinase 2; JUNK1, Jun N-terminal kinase 1; MAPK, MAP kinase; NEMO, NF-κB essential modulator; NIK, NF-κB-inducing kinase; IKB, inhibitors of NF-κB; IKK, IκB kinases; ERK, extracellular signal-regulated kinase; JNK, Jun N-terminal kinase; AP-1, activator protein; TBP, TATA-binding protein.

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