There was a significant positive statistical correlation between the panel of (NF-κB and bFGF) staining and number of layers of synovial cells and lymphocytic aggregates. Furthermore, bFGF correlated significantly with perivascular lymphocytic infiltrate (Table 6).
Findings and interpretation
In this work, we observed middle aged female predominance among RA patients. There was a significant statistical correlation between the patient and the control group in elevated ESR and the presence of microcytic hypochromic anemia. Histological examination showed pannus formation together with fibrosis, increased vascular density, and dense lymphocytic infiltration in the patient group. Immunohistochemical staining panel for NF-κB and bFGF showed a strong statistically positive correlation with the RA group. These findings might focus our interest to these strong associations as NF-κB and bFGF might play a very active role in the initiation, augmentation, and maintenance of pannus formation.
The histological findings of fibrosis and pannus formation could be attributed to the synovial fibroblast-like synoviocytes (FLS). These FLS are known as pannocytes. They are prominent at the interface between the synovium and the cartilage, leading to invasion and destruction of the adjacent cartilage, even in the absence of inflammatory infiltrate (Longato et al., 2005).
The abnormal behavior of FLS in inflammatory arthritis might be the result of spontaneous mutations leading to dysregulation of FLS function as NF-κB, which increases the aggressiveness of proliferating FLS. Another driving force for autonomously activated and the transformed nature of FLS results from continuous stimulation by inflammatory mediators and growth factors, such as bFGF, leading to expansive and tumor-like synovial pannus that invades cartilage (Guo et al., 2012).
This pannus requires additional nutrients and oxygen. In the face of this demand, there is likely a shift in the balance between angiogenic and antiangiogenic factors, with a predominance of the angiogenic mediators, such as bFGF, leading to neovascularization (Rudolph and Woods, 2005).
Although the RA specimens showed increased vascular density in comparison with the control group (P<0.001), they lacked a correlation with bFGF staining; this might be because of excessive specimen fibrosis and nonusage of specific vascular endothelial immunohistochemical markers in our research.
Differences in results and conclusion in comparison with other studies
In terms of histopathological findings, our results showed that there was a statistically highly significant increase in the mean number of synovial cells and vascular density in RA patients (5.2±2.5 vs. 1.1±1.1 and 5.7±1.1 vs. 1.5±0.7, respectively; P<0.001).
This was in agreement with Roccaro et al. (2005), who added that neovascularization in RA may be caused by hypoxia of the hyperplastic synovium and the cytokine milieu, which stimulate the release of angiogenic factors such as bFGF, VEGF, angiopontin, and fractalkine.
In our study, we found a statistically highly significant increase in the mean number of free lymphocytes between RA patients (2.8±1.3) and control individuals (0.45±0.4) as P value less than 0.001.
Our results were in agreement with those of Fonseca et al. (2000) who found that the presence of lymphocytes was statistically significant in RA synovial samples. Other authors have reported that T lymphocytes play an important role in the pathogenesis of RA (Remans et al., 2005).
In our study, we detected a highly significant immunohistochemical expression of nuclear NF-κB in the synovial biopsy of RA patients compared with the control group in synovial lining, sublining, and endothelial cells (84 vs. 10%, 60 vs. 0%, and 76 vs. 0%,with P<0.001, P<0.05, and P<0.001), respectively.
Our results were in agreement with those of Handel et al. (1995), who identified p50 and p65 subunits of NF-κB in the nuclei of cells within 100% of the lining layer, 69.2% of sublining region, and 84.6% of endothelial cells of RA synovium.
NF-κB is activated and increased in the tissue as a result of local hypoxia, oxygen-free radicals, and many inflammatory cytokines. Many trials are ongoing for the treatment of many diseases, such as RA, by inhibiting NF-κB (Kannaiyan et al., 2011a, 2011b; Li et al., 2012).
Moreover, Kubota et al. (2007) reported the importance of inhibiting NF-κB to relieve the inflammation and bone destruction in an animal model of arthritis as it acted as a transcription factor implicated in diverse receptor-mediated signaling pathways including the differentiation and activation of osteoclasts.
In this study, we found a highly significant correlation between the panel expression of NF-κB in the lining synovium, sublining cells, endothelial cells with the mean number of synovial cell layers, and lymphocytic aggregates as P value less than 0.01.
This was in agreement with Benito et al. (2004) who reported that the NF-κB signaling pathway was a driving factor for the hyperplastic changes in the synovium; also, it acted as a key mediator of inflammation, which is of critical value in the survival of synovial fibroblasts in RA joints.
In our study, there were topographic differences in the distribution of bFGF staining; it was detected in the nuclei and cytoplasm of the synoviocytes of RA synovial specimens, whereas it showed only cytoplasmic stain in the control group. Moreover, bFGF was positively stained in fibroblasts, and endothelial cells in RA patients, and totally negative in the control group.
These results were in agreement with those of Nakashima et al. (1994), who detected bFGF immunohistochemically in the cytoplasm and/or nucleus of the synovial lining cells of all RA synovial specimens, 86% of fibroblasts, and 73.3% of endothelial cells, and they found no expression of bFGF in the synovial tissue sections of five normal control individuals with joint trauma.
Yamashita et al. (2002) and Nakano et al. (2004) found that bFGF not only augments the proliferation of rheumatoid synovial fibroblasts but is also involved in osteoclast maturation, which leads to bone destruction in RA.
In our study, we found that there was a statistically highly significant positive correlation between staining of bFGF in synoviocytes, fibroblasts, and endothelial cells with the mean number of synovial cell layers, perivascular lymphocytic infiltration, and lymphoid aggregates.
These findings were in agreement with Roccaro et al. (2005), who found in their study that although perivascular mononuclear cell infiltration and thickness of synovium were increased in both inflamed and noninflamed joints, vascular proliferation occurred only in tissues from inflamed joints. The endothelial cells of these proliferating vessels were shown to express cell-cycle-associated antigens, such as PCNA and Ki-67, which are usually associated with vascular proliferation.
Relevance of the findings: implications for clinicians
NF-κB and bFGF inhibitors may be a potentially important therapeutic approach for RA by correcting the imbalance between apoptosis and proliferation of synovial cells, on the one hand, and angiogenesis and antiangiogenesis of the synovial tissue, on the other, in RA patients.
From this study, we can conclude that NF-κB and bFGF play a crucial role in the pathogenesis of rheumatoid pannus by inducing synovial hyperplasia and angiogenesis. Inhibition of NF-κB and bFGF may represent a new tool for future therapeutic strategies.
Conflicts of interest
There are no conflicts of interest.
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©2013Egyptian Journal of Pathology
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