Direct in vivo evidence of activated macrophages in human osteoarthritis

Abstract

Objective: Through binding to folate receptor-β (FR-β), the new (99m)Tc-EC20 (Etarfolatide) imaging technique detects activated but not resting macrophages in vivo. The goal of this study was to investigate macrophage-related inflammation in osteoarthritis (OA).

Methods: Twenty-five individuals (50 knees) with symptomatic OA of at least one knee underwent SPECT-CT imaging of both knees and planar imaging of the whole body after injection of Etarfolatide. Scans and knee radiographs were scored blinded to clinical information including knee and other joint site pain severity. Measures of association controlled for age, gender, body mass index (BMI) and employed repeated measures to adjust for correlation between knees.

Design: Activated macrophages were present in the majority (76%) of knees. The quantity of knee-related macrophages was significantly associated with knee pain severity (R = 0.60, P < 0.0001) and radiographic knee OA severity including joint space narrowing (R = 0.68, P = 0.007), and osteophyte (R = 0.66, P = 0.001). Macrophages were also localized to joints commonly affected by OA including hand finger joints (12%), thumb bases (28%), shoulders (26%), great toes (18%) and ankles (12%). The presence of joint pain at fingers, wrists, ankles and great toes was significantly positively associated with presence of activated macrophages at these sites (P < 0.0001-0.04).

Conclusions: This study provides the first direct in vivo evidence for macrophage involvement in OA in a substantial proportion of human knees. The association of quantity of activated macrophages with radiographic knee OA severity and joint symptoms suggests that drugs targeting macrophages and macrophage-associated inflammatory pathways may have the potential to be both symptom and structure modifying.

Trial registration: ClinicalTrials.gov NCT01237405.

Keywords: Inflammation; Joint pain; Knee; Macrophage; Osteoarthritis.

Conflict of interest statement

All authors met all criteria for authorship in the ICMJE Recommendations. Virginia B Kraus designed the study and supervised all aspects of the study conduct, interpretation and reporting. Gary McDaniel, PAC served as the study coordinator under the supervision of Dr. Kraus for all aspects of the study. Janet L Huebner assisted with study logistics and performed, sample handling and study monitoring. Thomas Stabler coordinated and performed all sample management. Carl F Pieper provided statistical support for the project. Steven Shipes served as the study coordinator for all radiological procedures and performed all the Etarfolatide imaging studies. Neil A. Petry actively participated in the imaging protocol design, IND preparation, study management and manuscript preparation and review process. He was also responsible for the procurement, compounding, quality control and dispensing of 99mTc–Etarfolatide imaging agent administration to subjects enrolled in this study. Philip S Low assisted with study design and interpretation. Dr. Low is the inventor of EC20 (Etarfolatide). Jiayin Shen performed the immunohistochemical staining for the synovial fluid samples and was an employee of Endocyte. Peter Mitchell, contributor to the study concept and design, and Terry A McNearney, contributor to the study design and logistics, are employees of Eli Lilly and Co.

Copyright © 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Figures

Fig 1. Structure of the EC20 imaging reagent

Folate, the targeting moiety, is shown on the left; the chelating agent to which 99mTc is conjugated, is shown on the right.

Fig 2. Consort Flow Diagram

Flow of participants through each stage of the observational study.

Fig 3. Representative patterns and intensity of Etarfolatide uptake in each of the 50 knees

Representative images (top row) of Etarfolatide uptake in the: A) joint 'capsule' (showing capsular and/or underlying synovial uptake, indicated in coronal view by arrowhead); B) 'synovium' (showing uptake by proliferative synovium in the posterior synovial recess, indicated in coronal and sagittal views by arrow); C) 'synovium' and joint 'capsule' (indicated in sagittal view by arrow and arrowhead, respectively); and D) bone (indicated in coronal view by asterisk). Heat map graphic (bottom) representing the uptake intensity for each knee as quantified for three regions: joint 'capsule', 'synovium' and subchondral bone; the maximum score for each joint region was 6, 6 and 5, respectively.

Fig 4. Knee radiographs, Etarfolatide scans and synovial fluid cell immunohistochemistry for knees of two study participants

In both participants, the left knee was the index knee. The panels on the left correspond to one participant and on the right to a second participant). Top: Bilateral anteroposterior knee radiographs show osteoarthritis bilaterally with more severe disease of the left knees for each participant; total osteoarthritis severity scores (joint space narrowing plus osteophyte) were 5, 9, 3 and 11 for right and left knees of participants 24 and 25 respectively. Corresponding pain scores were 1, 3, 1 and 3 respectively. Middle: The total Etarfolatide knee scores were 3, 5, 0 and 9 respectively (the second row shows SPECT images and the third row shows matched fused SPECT-CT images of the knee; coronal and transaxial images include both right and left knees, and the sagittal images include the index left knees of each participant. Synovial fluid cytospins were stained for iNOS, FR-β and TGF-β. Merged images demonstrate co-expression of FR-β (identifying the cells as activated macrophages), an M1 macrophage marker (iNOS) and an M2 macrophage marker (TGF-β). R=right knee; L=left knee.