CD68+ macrophages as crucial components of the foreign body reaction demonstrate an unconventional pattern of functional markers quantified by analysis with double fluorescence staining

Abstract

Implants like meshes for the reinforcement of tissues implement the formation of a persistent inflammation with an ambient fibrotic reaction. In the inflammatory infiltrate several distinct cell types have been identified, but CD68+ macrophages are supposed to be most important. To investigate the collaboration among the various cell types within the infiltrate we performed at explanted meshes from humans double fluorescence staining with CD68 as a constant marker and a variety of other antibodies as the second marker. The list of second markers includes lymphocytes (CD3, CD4, CD8, CD16, CD56, FoxP3, and CD11b) stem cells (CD34), leucocytes (CD45, CD15), macrophages (CD86, CD105, CD163, and CD206); deposition of EC matrix (collagen-I, collagen-III, MMP2, and MMP8); Ki67 as a marker for proliferation; and the tyrosine-protein kinase receptor AXL. The present study demonstrates within the inflammatory infiltrate the abundant capability of CD68+ cells to co-express a huge variety of other markers, including those of lymphocytes, varying between 5 and 83% of investigated cells. The observation of co-staining was not restricted to a specific polymer but was seen with polypropylene fibers as well as with fibers made of polyvinylidene fluoride, although with differences in co-expression rates. The persisting variability of these cells without the functional reduction toward differentiated mature cell types may favor the lack of healing at the interface of meshes.

Quantitative Characterization of Macrophage, Lymphocyte, and Neutrophil Subtypes Within the Foreign Body Granuloma of Human Mesh Explants by 5-Marker Multiplex Fluorescence Microscopy

“…Any quantification needs to define a reliable cut-off to determine at what intensity a cell may be considered “positive” ( 9 ). The cut-off usually does not result from natural order with distinct intensities but always has to be fixed manually and arbitrarily ( 10 ). The intensity may be measured as mean or maximum in an eroded or dilated nuclear mask.…”

Tissue remodeling macrophages morphologically dominate at the interface of polypropylene surgical meshes in the human abdomen

“…For cells, the analysis of the staining intensity only in the area of the nuclei cannot exclude positive staining based on some overlapping cytoplasm membrane, but the probability is considerably reduced by sectioning at a thickness of about 2 µm. In addition, we applied a cutoff value of 100 for the mean staining intensity of cells, as described [ 13 ]. This allowed us to determine positive cells in an objective and reliable way and provided us with the most confidence for true positive detection.…”

“…The Euclidean distance is a method commonly used in image processing that uses the Pythagorean formula to measure the distance between two pixels in a straight line. The detection of cells and positive marker signals was done as described previously [ 13 ]. Briefly, optimized DAPI images were used to detect and segment nuclei whose areas were used to measure the mean staining intensities for FITC- and Cy5-shades of the respective markers.…”

Characterization of innate and adaptive immune cells involved in the foreign body reaction to polypropylene meshes in the human abdomen

“…Previously, we reported that CD68 is co-expressed on various cell types participating in the FBR to meshes, which is considered a “hybrid” pattern in contrast to “conventional” cells expressing their specific surface markers without CD68 [ 19 ]. Subsequently, we showed that tissue remodeling macrophages dominate at the interface of fibrotic PP meshes in the human abdomen that have been removed for the primary complaint of recurrence [ 20 ].…”