Immune mediated disturbances of bone, connective tissue and vascular metabolism in Complex Regional Pain Syndrome (CRPS) : a new pathogenic mechanism of therapeutic relevance
CRPS is a neuropathic pain syndrome with severe trophic disturbances, which develops in about 0.2 - 0.5% of all trauma patients following a limb trauma or surgery. In Germany with about 4000-8000 new cases expected per year, about 30-40% of patients show a chronic course, often leading to loss of ability to use the affected limb. The main symptoms include a severe neuropathic pain syndrome, accompanied by severe trophic disturbances such as edema, increased skin temperature, impaired regulation of blood flow and osteoporosis. These autonomic / trophic changes indicate disturbances in the vascular regulation, the connective tissue and bone metabolism. Although neurogenic inflammation in the affected limb is an important factor of the disease, the actual trigger factors, in addition to the trauma, are not yet known. As a new factor we were able to show autoimmune process against autonomic nerve fibers as well as against neuronal cell surface epitopes. This includes functionally active autoantibodies against the ADRB2 (beta2 adrenergic receptor) and CHRM2 (muscarinic M2 receptor). An expression of our identified target receptors (ADRB2 and CHRM2) on endothelial cells, smooth muscle, osteosarcoma cells and osteoblasts have been described in previous literature and confirmed by us through the expression studies. Initially, sera obtained from CRPS patients showed higher binding to the CHO-cells overexpressing the ADRB2 and CHRM2 receptors. Blocking the target receptors with their specific antagonists reversed the binding effects. As our main interest in this study is to address the inflammatory mechanisms in vascular endothelial cells and bone related issues in CRPS, we did further detailed study with HCMEC cells and primary osteoblasts. We were able to find that 86% of patients showed effective binding to the target receptors expressed in endothelial HCMEC cells. Binding of these autoantibodies lead to higher cytotoxic effects in the HCMEC cells and decreased proliferation rate compared to controls. Additionally, expression studies proved that CRPS IgG incubation to vascular endothelial cells stimulated highly elevated levels of pro-inflammatory mediators and adhesion molecules such as ICAM-1, VCAM-1 and MCP-1. We were also able to show that CRPS IgG is a potent stimulator of ERK 1/2, P38 MAPK and STAT-1 pathways. TGF-Epsilon 1 (transforming growth factor, a key regulator of cell growth, cell proliferation, cell differentiation) and BMP-2 (bone morphogenic protein, key regulator of TGF beta signaling pathway) levels were reduced which could be a possible explanation for less proliferation rate of HCMEC cells. This was confirmed by the reduced levels of AKT phosphorylation in HCMEC cells that were stimulated with CRPS IgG.Secondly, our results showed that CRPS IgG binding to primary osteoblasts from different patients varies in their binding, cytotoxic and proliferation properties. Surprisingly, all three different osteoblasts incubated with CRPS IgG showed higher necrosis but not apoptosis, which explains that these IgG has damaging pathogenic effects on the bone cells.Additionally, we were also able to find that CRPS IgG incubated for 24hrs with the primary osteoblasts lead to reduced expression levels of osteoblast-specific markers such as Col-1, DKK-1, ALP and BGLAP/osteocalcin. All these markers are essential for bone mineralization and differentiation process. Reduced expression of these markers explains a possibility for poor bone formation and osteoporotic events in these patients.Finally, we also studied the mechanism of the immunomodulator IvIg on the CRPS IgGinduced effects on HCMEC, HEK293 and A10 cells and observed CRPS IgG when incubated along with IvIg had reduced cytotoxic effects in these cells. Although there was not much improvement were observed in their proliferation rates. Therefore, IvIg if studied in detail about their mechanism of action, can be used as a potent drug for treating CRPS. One possible mechanism may be the occurrence of idiotypic antibodies in the IvIg preparations as described in the treatment of other autoimmune diseases. The detection of functionally active autoantibodies in serum samples from CRPS patients against autonomic nervous structures, adrenergic and muscarinic receptor suggests an autoimmune participation as an important pathogenic factor in the pathophysiology of CRPS. Thus, our findings contribute to an understanding of the molecular mechanisms that are involved in inflammation and local trophic changes and possibly drive a way for new treatment strategies focusing on the immune system.
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