Aleem A Khan

Avinash Bardia

Dr Aleem A Khan

G Lakshmi Lavanya

M Phanibhushan

Mazaharuddin A Khan

S Vishnupriya

Sandeep K Vishwakarma

Santosh K Tiwari

CiteSeerX

ISSN 2320-5407                             International Journal of Advanced Research (2013), Volume 1, Issue 10, 139-144 
 
139 
 
                                                   Journal homepage: http://www.journalijar.com                 INTERNATIONAL JOURNAL 
                                                                                                                           OF ADVANCED RESEARCH 
                                                                                                                               
RESEARCH ARTICLE 
 
Monocyte Migration and Inflammatory Response in the Pathogenesis of 
Rheumatoid Arthritis 
 
Mazaharuddin A khan,
3
 Avinash Bardia,
1
 Santosh K. Tiwari,
1
 Sandeep K Vishwakarma,
1 
M. 
Phanibhushan,
2
 G. Lakshmi Lavanya,
2
 S. Vishnupriya,
2
 Aleem A. Khan,
1* 
1. Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad, INDIA. 
2. Department of Genetics, Osmania University, Hyderabad, AP, INDIA. 
3. Department of Genetics, Osmania University, Hyderabad, AP, INDIA. 
 
Manuscript Info                  Abstract  
 
Manuscript History: 
 
Received: 12 November 2013 
Final Accepted: 28 November 2013 
Published Online: December 2013                                          
 
 
 
*Corresponding Author 
 
Dr. Aleem A. Khan 
Centre for Liver Research and 
Diagnostics, Deccan College of 
Medical Sciences, Hyderabad, 
INDIA. 
Ph./Fax: +91-040-24342954 
aleem_a_khan@rediffmail.com 
 
 
 
Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily 
affects multiple synovial joints. Monocytes/Macrophages accumulate in 
synovium, where they play a central role in inflammation and joint 
destruction. In human peripheral blood, two monocyte subpopulations with 
distinct functional properties have been defined by their expression of CD14 
and CD16 molecules. In the present study, we investigated the relevance of 
CD14+, CD16+, MIF and TLR4 expression in blood monocytes in RA 
population. Flowcytometric analysis was done for different monocytes by 
staining with CD14, CD16, MIF and TLR4 tagged with flurochromes in 
order to assay the inflammatory cytokines from 257 RA patients and 270 
healthy controls. Inflammatory monocytes (CD14+ and CD16+) population 
was significantly high in subjects with RA than the healthy control. mCD14, 
mTLR4, CD16 and MIF expression levels were also increased the 
susceptibility of RA. The study also found that expression level of TLR4, 
CD14, MIF and CD16 can be used as a diagnostic biomarker for RA. 
 
                   Copy Right, IJAR, 2013,. All rights reserved.
 
Introduction  
 
 
Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects multiple synovial joints. Synovial 
infiltration with cells of the monocyte/macrophage lineage is thought to be responsible for both the chronic 
inflammation and the joint destruction (Burmester, et al. 1997). Of importance, activated macrophages are the major 
source of many cytokines in the joint, including tumor necrosis factor α (TNF α), IL-1, IL-8, and granulocyte–
macrophage colony-stimulating factor (Feldmann, et al. 1996). These cytokines are intimately involved in the 
disease process. In particular, the significance of TNF α in RA has been well proven by the clinical efficacy of TNF 
α blockade in patients with active disease (Elliott, et al. 1993; Moreland, et al. 1997). The activation of blood 
monocytes before entry into the joint in RA has previously been documented. PB monocytes from RA patients, 
compared with monocytes from healthy individuals, express higher levels of adhesion molecules (Liote, et al. 1996; 
Shinohara, et al. 1992) and show increased phagocytic activity (Steven, et al. 1984). In addition, RA monocytes are 
able to spontaneously produce inflammatory molecules, including cytokines such as TNFα, IL-1, and IL-6 (Shore, et 
al. 1986; Goto, et al. 1990; Stuhlmuller, et al. 2000), the arachidonic acid metabolites (Seitz, et al. 1982), 
complement components (Ceulaer, et al. 1980), and angiotensin-converting enzyme (Goto, et al. 1990). 
Furthermore, both quantitative and qualitative alterations of myelomonocytic lineage cells in the bone marrow have 
been reported (Ochi, et al. 1988; Hirohata, et al. 1995; Tomita, et al. 1997). These findings strongly suggest that the 
monocyte/macrophage system is activated in RA. Macrophages are principal drivers of synovial inflammation in 
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RA, a prototype immune-mediated inflammatory disease. Monocytes/macrophages accumulate in synovium, where 
they play a central role in inflammation and joint destruction. Interleukin (IL) and tumour necrosis factor (TNF) 
alpha are pleiotropic cytokines produced predominantly by macrophages which have been implicated in the 
pathogenesis of RA. In human peripheral blood, two monocyte subpopulations with distinct functional properties 
have been defined by their expression of CD14 and CD16 molecules. The cytokine macrophage migration inhibitory 
factor (MIF), CD14, and Toll-like receptor 4 (TLR4) are interlinked components with clearly defined roles in 
immunologic and inflammatory pathways (Calandra, 2003). MIF regulates innate immune responses through the 
modulation of pattern recognition receptors like TLR4 (Roger, et al. 2001). TLR4, a signal-transducing receptor, is 
mainly responsible for the recognition of bacterial lipopolysaccharide. In brief, TLR4 specifically associates with 
lymphocyte antigen 96 (MD2) and, together with CD14, forms a lipopolysaccharide complex that aides in 
lipopolysaccharide recognition and nuclear factor NFκB activation (Calvano, et al. 2003). So the present study is 
designed to investigate the expression of CD14, CD16, TLR4 and MIF in blood monocytes in RA.  
 
Materials & Methods  
 
Study population and sampling  
The study population consisted of 527subjects, 257 with active RA and 270 healthy controls. The RA group was 
composed of only patients with a documented history and having a current episode, whereas the healthy control 
group included subjects without any history of RA or any other autoimmune disorder. Written and signed informed 
consent was obtained from all patients. The study protocol and procedures were approved by the Institutional Ethics 
Committee, Deccan College of Medical Sciences (Hyderabad, India).  
A total of 6 mL peripheral venous blood (2 mL in plane tube for serum separation and 3 mL in heparinized 
vacutainer for mononuclear cells isolation) was collected. Mononuclear cells were isolated using Histopaque (Ficoll-
paque) solution according to the manufacturer’s instructions.  
 
Flow cytometric analysis  
 
Expression levels of CD14, CD16, TLR4 and intracellular MIF  
Purified mononuclear cells were diluted with phosphate buffered saline (pH 7.0) at 5 x 10^6 cells/mL (containing 
10–25% monocytes). Specimen processing and antibody staining were performed according to the manufacturer’s 
instructions. For CD14 levels, PE-conjugated antibodies against CD14 (BD Biosciences, San Jose, CA) were 
incubated with peripheral blood mononuclear cells (PBMCs). For TLR4, PBMCs were incubated with a PE-
conjugated antihuman TLR4 (clone HTA125) or mouse immunoglobulin G2a as isotype control (eBioscience, San 
Diego, CA). For the detection of intracellular TLR4 levels, cells were permeabilized with permeabilization buffer 
(eBioscience) and stained with antihuman TLR4 antibody or relevant isotype. For CD16 levels, FITC-conjugated 
antibodies against CD16 (BD Pharmingen, San Jose, CA) were incubated with PBMCs. Similarly, intracellular MIF 
expression was evaluated using anti-MIF monoclonal antibody (mAb; R&D Systems, Minneapolis, MN). In brief, 
freshly isolated PBMCs were incubated with anti-MIF mAb and secondary mAb conjugated with FITC. Data 
acquisition and analysis were performed using CellQuest software on a BD Calibur flow cytometer (BD 
Biosciences) that automatically and simultaneously measured the fluorescence of individual cells identified by their 
size-dependent light-scattering properties.  
 
Statistical analysis 
  
Expression of CD14, TLR4, CD16 and MIF were analyzed using one-way analysis of variance followed by 
Newman–Keuls multiple comparison test to determine the significance and difference between the various groups. 
Analysis was performed using GraphPad Prism software (version V, GraphPad software, Inc., CA).  
 
Results  
 
Expression of CD14, TLR4, CD16 and intracellular MIF was measured in terms of mean fluorescent intensity 
(MFI). In CD14 expression MFI demonstrated a significant variation between RA subjects and healthy subjects 
(p<0.05; patients vs controls). A significant difference in MFI was also recorded between RA patients and healthy 
controls in TLR4 expression. The highest levels of CD16 expression were observed in subjects with RA when 
compared with healthy subjects. MIF expression levels indicated a significant difference between patients and 
healthy controls. 
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Figure 1: Histogram plot illustrating the differential expression of CD14 rheumatoid arthritis 
patients and healthy controls 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2: CD14 expression levels differed in 
rheumatoid arthritis patients compared with 
control subjects. **p 0.005 
Figure 3: Histogram plot illustrating the 
differential expression of CD16 
rheumatoid arthritis patients and 
healthy controls 
Figure 4 CD16 expression levels 
differed in rheumatoid arthritis 
patients compared with control 
subjects. **p 0.005 
Figure 5: Histogram plot illustrating 
the differential expression of TLR4 
rheumatoid arthritis patients and 
healthy controls 
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Discussion 
  
CD14+, CD16+ monocytes are a subpopulation of cells that while in the circulation, has acquired features in 
common with mature tissue macrophages.CD16+ monocytes can rapidly migrate to the site of inflammation, where 
they readily mature into proinflammatory macrophages. CD14, TLR4, and MIF genes represent important and 
intertwined determinants of immunity. A well-orchestrated and synchronized interaction between these is highly 
essential for an optimal first line of defense (Admetlla, et al. 2008). Alteration in this normal process significantly 
dislodges the immunity network, resulting in immunologic imbalance. RA is one such paradigm of immune 
dysregulationas a result of persistent inflammation. We studied the effects of CD14, CD16, TLR4, and MIF on 
respective mononuclear cells expression levels in patients with RA. In recent years, there has been large influx of 
expression studies on different genes that are either directly or indirectly involved in the disease process. Because 
MIF, CD14, and TLR4 genes represent a triad that regulates innate immunity; the present study attempted to 
demonstrate the significance of expression in candidate genes (CD14, CD16, MIF and TLR4) of innate immunity in 
increasing the risk of RA. This study demonstrated the effect expression patterns of their respective proteins. As can 
be clearly seen from the data the expression of CD14 and CD 16 differed significantly between patients and controls 
(Fig 1, 2 and 3, 4).A similar trend was observed with TLR4 expression levels (Fig 5 and6). Kawanaka et al 
(Kawanaka, et al. 2000) suggested that the numbers of CD14+, CD16+ blood monocytes increase during the active 
phase of RA which supports our data. An important role for the CD16 molecule induced on monocytes in RA has 
recently been indicated by Abrahams et al (Abrahams, et al. 2000). Since MIF was first identified in 1966, its 
influence on the pathogenesis of many diseases, including RA, has been investigated extensively (Bloom, et al. 
1996). Many studies have shown that MIF plays a pro-inflammatory role in many inflammatory diseases, such as 
sepsis (Bernhagen, et al. 1993), RA (Leech,  et al. 1998), delayed-type hypersensitivity (Bernhagen, et al. 1996), 
glomerulonephritis (Lan, et al. 1997), and various tumors (Nishihira, et al. 2003). In our study a similar trend was 
observed with MIF expression levels (Fig 7).The link between increased TLR4 surface expression and MIF cytokine 
production is compelling since several lines of evidence are emerging that suggest a regulatory relationship between 
the two.In conclusion, this study demonstrated that the CD14, CD16, TLR4 and MIF gene modulate their expression 
levels on monocytes and also confer a greater risk of developing RA. Because these genes are interlinked with each 
other, further studies are required to gain insight how each of the genes alter the protein structure and interaction, 
resulting in perturbation of the delicate immunologic balance in RA.  
 
Acknowledgement: None 
 
Conflict of interest: None 
Figure 6: TLR4 expression levels 
differed in rheumatoid arthritis patients 
compared with control subjects.  
**p 0.005 
Figure 7: Intercellular MIF expression 
levels differed in rheumatoid arthritis 
patients compared with control subjects. 
**p 0.005 
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Monocyte Migration and Inflammatory Response in the Pathogenesis of Rheumatoid Arthritis

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Monocyte Migration and Inflammatory Response in the Pathogenesis of Rheumatoid Arthritis

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