Abstract
Current treatments for cardiovascular diseases use biological implants to replace or repair nonfunctional tissue. Besides a low immunogenicity, those matrices should induce balanced immune responses enabling proper tissue regeneration. Polymorphonuclear leukocytes (PMNs) represent the first cellular players of an immune response, initiating subsequent attraction and activation of other immune cells. However, so far, robust in vitro assays are missing that predict PMN responses to diverse implant matrices and help to understand immune response mechanism. Therefore, we aimed to establish an easy and standardized in vitro assay to assess matrix-induced PMN responses. First, PMNs were isolated from blood of healthy donors and stimulated with various cytokines, chemokines as well as lipopolysaccharide (LPS) and the peptide N-formyl-met-leu-phe (fMLP). To select appropriate activation conditions and markers, surface expression of CD11b, CD16b, CD35, CD43, CD62L, CD63, and CD66b on PMNs was determined by flow cytometry after 4 h in culture. In addition, the cytokine secretion of interleukin (IL)-6, MIP-1β, and IL-8 was measured by enzyme-linked immunosorbent assays (ELISA). Second, PMNs were cultured on “proof-of-principle” matrices: (1) aortic tissue after conventional frozen cryopreservation or ice-free cryopreservation and (2) decellularized amniotic membrane (DeAM), additionally coated with a cardiac extracellular matrix hydrogel (DeAM+E), and analyzed for PMN activation. The activation assay validated LPS as most effective and dose-dependent inducer of PMN activation in vitro. The increase of CD63 and CD66b expression and decrease of CD16b, CD43, and CD62L on PMNs, as well as enhanced IL-6 secretion, were determined as reliable PMN activation markers. Both cryopreserved matrices did not trigger any PMN activation. In contrast, DeAM+E, but not DeAM alone, induced a similar expression level for CD43, CD62L, and CD63 on PMNs as the LPS activation control, but no increase of IL-6 secretion. In this study, we developed an easy, fast, and suitable assay to measure the PMN activation by biological matrices in vitro through a characteristic activation marker and cytokine profile to reveal mechanism of their potential immunogenicity and compatibility when used as implants. The established PMN assay could be a powerful additional tool to existing in vitro tests and might be implemented in the evaluation for future clinical implants.
Impact Statement
Polymorphonuclear leukocytes (PMNs) are essential in the first infection and host-versus-graft reactions. Strategies for adequate and standardized assays to test PMN activation by diverse types of matrices such as cardiovascular implants are urgently needed. To overcome this limitation, we established a straightforward PMN activation assay and validated lipopolysaccharide (LPS) as a reliable PMN activator that induces defined changes in surface marker expression and cytokine release. Biological “proof-of-principle” matrices demonstrated the feasibility of this PMN assay. Overall, this assay provides an instrument conducting an initial immunological assessment of biological implants prior their clinical application.
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