The molecular pathophysiology of subacromial bursitis in rotator cuff disease

The molecular pathophysiology of subacromial bursitis in rotator cuff disease Theodore A. Blaine, MD,a,b Yang-Soo Kim, MD,a,b Ilya Voloshin, MD,a,b Darwin Chena,b, Koko Murakami, PhD,a,b Seong-Sil Chang, PhD,a,b Robert Winchester, MD,a,b Francis Y. Lee, MD, PhD,a,b Regis J. O’Keefe, MD, PhD,c and Louis U. Bigliani, MD,a,b

Little information exists on the molecular and biochemical pathophysiology of subacromial bursitis and rotator cuff disease. We investigated the pattern of expression of cytokines (interleukin [IL]–1␤, IL-1, IL-6, tumor necrosis factor [TNF] ␣, small inducible cytokines), metalloproteases, and cyclooxygenases in the subacromial bursa in patients with rotator cuff disease. Subacromial bursa specimens were prepared for molecular and biochemical analysis in patients undergoing shoulder surgery following an institutional review board–approved protocol. Specimens were analyzed for the presence of cytokines, metalloproteases, and cyclooxygenases by use of microarray for gene expression and immunohistocytochemistry. Microarray analysis for gene expression and immunohistochemistry demonstrated that the expression of several cytokine genes (TNF, IL-1␣, IL-1␤, and IL-6) was increased in patients with subacromial bursitis compared with control specimens. Furthermore, the expression of metalloproteases (MMP-1 and MMP-9) and cyclooxygenases (COX-1 and COX-2) in the bursitis group was found to be increased as compared with controls. Although further investigation is required, these studies suggest that inflammation of the subacromial bursa does occur in patients with rotator cuff disease. These findings support the role of anti-inflammatory agents in the treatment of subacromial impingement and emphasize the importance of subacromial bursectomy to re-

From the aCenter for Orthopaedic Research and bCenter for Shoulder, Elbow and Sports Medicine, Columbia University, New York, and cMusculoskeletal Research Laboratory, University of Rochester, Rochester. Reprint requests: Theodore A. Blaine, MD, Assistant Professor of Orthopaedic Surgery, Co-Director, Columbia University Center for Orthopaedic Research, Associate Director, Center for Shoulder, Elbow and Sports Medicine, Columbia University College of Physicians and Surgeons, 622 W 168th St, PH 1120, New York NY 10032 (E-mail: [email protected]). Copyright © 2005 by Journal of Shoulder and Elbow Surgery Board of Trustees. 1058-2746/2005/$30.00 doi:10.1016/j.jse.2004.09.022

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duce inflammation in rotator cuff disease. (J Shoulder Elbow Surg 2005;14:84S-89S.)

In

1934 Codman1 claimed that the subacromial bursa “itself is the largest in the body and the most complicated in structure and in its component parts. It is in fact, a secondary scapulohumeral joint, although no part of its surface is cartilage.” Codman, therefore, gave significant functional purpose to the subacromial bursa. Neer8,9 further emphasized this concept in his characterization of subacromial impingement syndrome in 1972. Despite these observations by the founding fathers of shoulder surgery, the subacromial bursa has received relatively little emphasis in the surgical treatment of rotator cuff disease. Multiple clinical investigations have supported the value of subacromial decompression, but the major emphasis has been on the bony decompression (acromioplasty) as the primary means of preventing impingement. Some authors have even recommended preservation and repair of the subacromial bursa to assist in rotator cuff healing.6,10,16 More recent reports, however, have suggested that the subacromial bursa is an inflammatory membrane that can lead to shoulder pain through stimulation of afferent nerve endings and their products.3,5 Clinical studies have indicated that bursectomy and rotator cuff repair without acromioplasty may have equivalent results to those with acromioplasty.2,7 These findings suggest that bursectomy alone may be successful in alleviating the pain associated with impingement. These retrospective clinical outcome studies support the hypothesis of the subacromial bursa as an important factor in subacromial pathology. However, the role of the bursa continues to be a source of debate. Several recent investigations have implicated the subacromial bursa as a major factor in subacromial impingement syndrome. Santavirta et al12 found a preponderance of CD-2 and CD-11b mononuclear cells in the subacromial bursa of patients with bursitis. Similarly, Yanagisawa et al17 demonstrated increased expression of vascular endothelial growth factor in patients with impingement, implying chronic

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Table I Immunohistochemical grading scale Grade

Fibrous tissue/fibrocytes

Synovium/ synoviocytes

0 1 2 3 4

No staining None or minimal staining (0%-20%) Minimal staining (0%-20%) Moderate staining (20%-50%) Abundant staining (⬎50%)

No staining Focal staining Diffuse staining Diffuse staining Diffuse staining

inflammation and increased vascularity. Finally, Soifer et al13 demonstrated the presence of neurologic elements in the subacromial space, and Gotoh et al3,5 related increased expression of substance P (a pain mediator) in the subacromial bursa in patients with shoulder pain with impingement. Although these studies implicate the subacromial bursa in the pathogenesis of impingement, the changes in cell biology and expression of biochemical mediators in impingement syndrome have not yet been identified. The present investigations were undertaken to determine the role of the subacromial bursa in rotator cuff disease, whether it is a pathologic or reparative tissue, and whether “bursitis” in fact represents an inflammatory process. With enhanced understanding of the molecular pathogenesis of subacromial bursitis, we may further understand our current treatments (nonsteroidal anti-inflammatory drugs [NSAIDs], corticosteroids, bursectomy) and may ultimately devise new treatment regimens for rotator cuff disease. Our hypothesis predicts that increased levels of inflammatory mediators (cytokines, metalloproteases, and cyclooxygenases) are secreted by the subacromial bursa in impingement syndrome and rotator cuff disease. MATERIALS AND METHODS Following an institutional review board–approved protocol, subacromial bursa biopsy specimens were obtained intraoperatively from patients undergoing shoulder surgery and were analyzed by use of several biochemical, molecular, and immunohistochemical (IHC) techniques for the expression of cytokines, matrix metalloproteases (MMPs), and cyclooxygenases (COX). Microarray analysis for gene expression

Analysis was performed for 14 patients with subacromial bursitis undergoing miniopen rotator cuff repair, and 4 patients undergoing surgery for shoulder instability were used as controls. Ribonucleic acid was extracted from the samples by use of the RNeasy Fibrous Tissue Mini Kit (Qiagen Inc., Valencia, CA) and stored at ⫺70°C. Complementary deoxyribonu-

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cleic acid–array hybridization was done by use of GEArray Q Series Kits (SuperArray Bioscience Corp., Frederick, MD) for inflammatory cytokine/receptor genes. The arrays were hybridized with biotin-dUTP– labeled complementary deoxyribonucleic acid probes. Images of the bursal specimens were obtained by chemiluminescent detection on x-ray films. Data analysis and normalization were accomplished by use of ScanAlyze (Stanford University, Palo Alto, CA) and GEArray Analyzer (Biomol GmbH, Hamburg, Germany) software. Statistical analysis was performed by use of group statistical analysis (Levene test for equality of means, 2-tailed t test with equal variances both assumed and not assumed, and Mann-Whitney U test). Statistical significance was considered present at P ⬍ .05. IHC analysis

IHC analysis was performed for 18 patients undergoing shoulder surgery. Patients were divided into two groups: group I comprised control subjects with no rotator cuff tears (RCTs) (n ⫽ 8) and group II comprised patients with RCTs (n ⫽ 10). Specimens stained with hematoxylin-eosin and IHC antibody (MMP-1, MMP-9, interleukin [IL]–1, IL-6, tumor necrosis factor [TNF] ␣, COX-1, and COX-2) were examined by 2 blinded observers using a histologic scale (Table I). Statistical analysis was performed with the Wilcoxon rank sum test. RESULTS Microarray analysis for gene expression

The expression of several cytokine genes (IL-1␣, IL-1␤, and IL-6) was increased in patients with subacromial bursitis compared with control specimens (P ⬍ .05) (Figures 1 and 2). TNF-␣ had a trend toward increased expression in bursitis patients, but the results were not statistically significant. The receptors for IL-1 (IL-1R2) and IL-6 (IL-6R) were increased compared with controls (P ⬍ .05), whereas the expression of IL-6 signal transducer (IL-6ST) was not. The receptor for TNF-␣ (TNFRSF-1) and its soluble factor (LTBR) had a trend toward increased expression, but this was not statistically significant. There was a trend toward increased expression of many of the small inducible cytokine (SCY) gene family in patients with bursitis as compared with controls. Some members of the small inducible cytokine subfamily genes (SCYA24, SCYA25, SCYB10, SCYB5, and SCYE1) were significantly increased in patients with subacromial bursitis and rotator cuff disease (P ⬍ .05). These cytokines have not been previously described in the subacromial bursa, and the implications of these increases are not yet known.

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Figure 1 Average relative intensity of expression of cytokines (TNF and interleukins) in subacromial bursa specimens.

Figure 2 Average relative intensity of expression of small inducible cytokines in subacromial bursa specimens.

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Figure 3 Immunohistochemistry for the presence of IL-1 and IL-6 in the subacromial bursa of a patient with impingement syndrome (group I) compared with control (group II). (Hematoxylin and eosin stain, original magnification ⫻ 100.)

Figure 4 Immunohistochemistry for the presence of TNF in the subacromial bursa of a patient with impingement syndrome (group I) compared with control (group II). (Hematoxylin and eosin stain, original magnification ⫻ 100.)

IHC analysis

IHC analysis of bursal specimens demonstrated increased staining for inflammatory cytokines (IL-1, IL-6, and TNF-␣) and proteinases (MMP-1 and MMP-9) in the RCT group as compared with controls (P ⬍ .05) (Figures 3, 4, 5, and 6). COX enzymes (COX-1 and COX-2) were also increased in group II (RCT) (P ⬍ .05) (Figures 6 and 7). In the majority of specimens, staining was most intense in the synoviocyte-type lining cells of the subacromial bursa (Figures 3, 4, 5, and 7). DISCUSSION Although inflammation in the subacromial bursa has been proposed as the mechanism leading to pain in patients with rotator cuff disease, the exact roles of cytokines and other proteins during this inflammatory process are currently unknown. Initial reports have provided conflicting data on the presence and role of the various cytokines and metalloproteases in the subacromial bursa.

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Figure 5 Immunohistochemistry for the presence of MMP-1 and MMP-9 in the subacromial bursa of a patient with impingement syndrome (group I) compared with control (group II). (Hematoxylin and eosin stain, original magnification ⫻ 100.)

One study by Tsuzaki et al15 isolated human tendon cells for the rotator cuff and treated them with recombinant human IL-1␤. Messenger ribonucleic acid (mRNA) was then isolated and polymerase chain reaction performed for the presence of cytokines. Interestingly, IL-1␤ induced the expression of MMP-1, MMP-3, MMP-13, IL-1, IL-6, and COX-2 in these cells, indicating that expression of inflammatory mediators could occur even without the presence of inflammatory cells (leukocytes, monocytes, and macrophages.) Gotoh et al4 also demonstrated increases in IL-1 and IL-1 receptor antagonist mRNA in subacromial bursa biopsy specimens from patients with rotator cuff disease as compared with controls (anterior instability patients). In this study the amount of IL-1 expression was also correlated with the amount of shoulder pain as assessed by visual analog scale. In a third study, Sakai et al11 also found increases in the frequency of IL-1 and TNF mRNA expression in the subacromial bursa of patients with RCTs as compared with controls (anterior instability patients). Finally, one contrasting study of 17 subacromial bursal biopsy specimens from patients with rotator cuff disease found increased expression of IL-6 and granulocyte-macrophage colony-stimulating factor mRNA, but TNF and IL-1 mRNA levels were undetectable.14 The present investigation was designed to determine which, if any, of these inflammatory molecules are expressed in patients with subacromial bursitis and rotator cuff disease. In these initial studies, gene expression as demonstrated by microarray analysis of cytokines (TNF, IL-1, IL-6, SCY) was found to be increased in patients with rotator cuff disease as compared with controls. IHC analysis of bursal specimens demonstrated increases in cytokines and metalloproteases, including IL-1, IL-6, TNF, COX-1, and

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Figure 6 Graphic representation of relative staining intensity of the various cytokines in the subacromial bursa of patients with impingement syndrome (group I) compared with control (group II).

Figure 7 Immunohistochemistry for the presence of COX-1 and COX-2 in the subacromial bursa of a patient with impingement syndrome (group I) compared with control (group II). (Hematoxylin and eosin stain, original magnification ⫻ 100.)

COX-2. These findings may have important implications with regard to the use of COX inhibitors (NSAIDs) for subacromial bursitis, although additional confirmatory studies are necessary. The IHC data also suggest that expression of these molecules occurs primarily in the synoviocyte-like bursal-lining cells.

These data provide initial indications that cytokines and metalloproteases play an important role in the pathology of subacromial bursitis. The pattern of cytokine expression, however, requires continued investigation and may be dependent on the section of bursal tissue and, in particular, the type of cell that is analyzed. Further investigation will emphasize the isolation and characterization of these genes in the bursal synoviocyte, which appears to be the pivotal cell in this process. Although further investigation is required, these studies suggest that inflammation of the subacromial bursa does occur in patients with rotator cuff disease. These findings support the role of anti-inflammatory medication (NSAIDs and corticosteroids) in the treatment of subacromial impingement and emphasize the importance of subacromial bursectomy to reduce inflammation in rotator cuff disease. We wish to acknowledge the contributions of Michael Maloney, MD, and Anand Murthi, MD, for their assistance in obtaining surgical specimens. We also acknowledge the technical assistance of Farnaz Yassae, MD, Faye Chen, PhD, Meelan Patel, Carey Nadell, and Albert Pendleton. REFERENCES

1. Codman EA. The shoulder, rupture of the supraspinatus tendon and other lesions in and about the subacromial bursa. Boston: Thomas Todd, 1934. 2. Goldberg BA, Lippett SB, Matsen FA. Improvement in comfort

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