Fibroblast activation and inflammation in frozen shoulder

Abstract

Introduction: Frozen shoulder is a common, fibro-proliferative disease characterised by the insidious onset of pain and progressively restricted range of shoulder movement. Despite the prevalence of this disease, there is limited understanding of the molecular mechanisms underpinning the pathogenesis of this debilitating disease. Previous studies have identified increased myofibroblast differentiation and proliferation, immune cell influx and dysregulated cytokine production. We hypothesised that subpopulations within the fibroblast compartment may take on an activated phenotype, thus initiating the inflammatory processes observed in frozen shoulder. Therefore, we sought to evaluate the presence and possible pathogenic role of known stromal activation proteins in Frozen shoulder.

Methods: Shoulder capsule samples were collected from 10 patients with idiopathic frozen shoulder and 10 patients undergoing shoulder stabilisation surgery. Fibroblast activation marker expression (CD248, CD146, VCAM and PDPN, FAP) was quantified using immunohistochemistry. Control and diseased fibroblasts were cultured for in vitro studies from capsule biopsies from instability and frozen shoulder surgeries, respectively. The inflammatory profile and effects of IL-1β upon diseased and control fibroblasts was assessed using ELISA, immunohistochemistry and qPCR.

Results: Immunohistochemistry demonstrated increased expression of fibroblast activation markers CD248, CD146, VCAM and PDPN in the frozen shoulder group compared with control (p < 0.05). Fibroblasts cultured from diseased capsule produced elevated levels of inflammatory protein (IL-6, IL-8 & CCL-20) in comparison to control fibroblasts. Exposing control fibroblasts to an inflammatory stimuli, (IL-1ß) significantly increased stromal activation marker transcript and protein expression (CD248, PDPN and VCAM).

Conclusions: These results show that fibroblasts have an activated phenotype in frozen shoulder and this is associated with inflammatory cytokine dysregulation. Furthermore, it supports the hypothesis that activated fibroblasts may be involved in regulating the inflammatory and fibrotic processes involved in this disease.

Fig 1. Fibroblast activation markers in shoulder capsule.

Representative images of and control capsule and frozen shoulder tissue sections stained with Haematoxylin and Eosin (x10 magnification, bar indicates 200μm) and antibodies against CD248, CD146, VCAM, PDPN, CD34, and FAP (x40). Isotype control in bottom right corner (x10 magnification, bar indicates 150μm).

Fig 2. Fibroblast activation marker expression in control capsule and frozen shoulder.

Graphs illustrate Modified Bonar scoring & % of cells positively stained for samples of human capsule biopsies for expression of CD248, CD146, VCAM, PDPN, CD34 and FAP, mean ± SEM, n  =  10 for control capsule, n  =  10 for frozen shoulder capsule. Modified Bonar scoring system depicts mean score per sample based on 10 high power fields. 0  =  no staining, 1  =  <10%, 2  =  10–20%, 3  =  >20% + ve staining of cells per high power field. *p < 0.05, **p < 0.01 (Students t-test).

Fig 3. Inflammatory cytokine production from capsule derived fibroblasts.

IL-6, CCL20 and IL-8 secretion from healthy capsule and diseased capsule derived fibroblasts, mean ± SEM, n = 5 *p < 0.05 (Students t-test).

Fig 4. Fibroblast activation markers are upregulated in shoulder capsule upon inflammatory stimulus and inflammatory protein in healthy fibroblasts following IL-1β (10ng/ml) stimulation.

(a) Stromal activation marker (CD248, PDPN, VCAM & CD146) mRNA expression following IL-1β stimulation of cultured healthy derived fibroblasts. Data represented 2^-ΔΔCT, normalized to GAPDH housekeeping control, mean ± SEM, n = 5. *p < 0.05, **p < 0.01 compared to control samples (Students t-test). (b) Representative images of healthy capsule derived fibroblasts following IL-1β stimulation. Cells stained with antibodies against CD248, PDPN, VCAM and CD146, image at x40 magnification (bar is 200μm). Isotype control in bottom left corner (x40).

Fig 5. Activated capsule fibroblasts drive proinflammatory cytokine production.

IL-6, CCL20 and IL-8 mRNA and protein expression following IL-1β stimulation. mean ± SEM, n = 5, *p < 0.05, **p < 0.01 compared to control samples (Students t-test).

Fig 6. Immunobiology of frozen shoulder.

Schematic illustration representing the functional interplay between the “activated” stromal fibroblast (CD248+, CD146+,VCAM+,PDPN+) (stromal compartment), infiltrating and resident immune cells (immune compartment) and downstream cytokine/alarmin dysregulation interacting to drive a proinflammatory phenotype with aberrant tissue fibrosis in the frozen shoulder joint capsule.