Poly(ethylene glycol)-containing hydrogels promote the release of primary granules from human blood-derived polymorphonuclear leukocytes

Abstract

Polymorphonuclear leukocytes (PMNs) are recruited to sites of injury and biomaterial implants. Once activated, PMNs can exocytose their granule subsets to recruit monocytes (MCs) and mediate MC/macrophage activation. We investigated the release of myeloperoxidase (MPO), a primary granule marker, and matrix metalloproteinase-9 (MMP-9), a tertiary granule marker, from human blood-derived PMNs cultured on poly(ethylene glycol) (PEG) hydrogels, polydimethylsiloxane (PDMS), tissue culture polystyrene (TCPS) and gelatin-PEG (GP) hydrogels, with and without the presence of the bacterial peptide formyl-Met-Leu-Phe. Supernatants from PMN cultures on PEG-containing hydrogels (i.e., PEG and GP hydrogels) had higher concentrations of MPO than those from PMN cultures on PDMS or TCPS at 2 hours. PMNs on all biomaterials released comparable levels of MMP-9 at 2 hours, indicating that PMNs cultured on PEG-containing hydrogels have different mechanisms of release for primary and tertiary granules. Src family kinases were involved in the release of MPO from PMNs cultured on PEG hydrogels, TCPS and GP hydrogels and in the release of MMP-9 from PMNs cultured on all four materials. The increased release of primary granules from PMNs on PEG-containing hydrogels did not significantly increase MC chemotaxis, indicating that additional co-effectors in the dynamic inflammatory milieu in vivo modulate PMN-mediated MC recruitment.

Biomaterials differentially regulate Src kinases and phosphoinositide 3-kinase-γ in polymorphonuclear leukocyte primary and tertiary granule release

“…1B). These data suggest that the enhanced primary granule release from PMNs cultured on PEG hydrogels is adhesion mediated as it coincides with the time frame of cell adhesion [17,39–41]. The MPO concentrations released into the supernatant at 2 h correspond to 10–22% of total MPO content for PMNs cultured on PEG and GP hydrogels and 1–4% of total MPO content for PMNs cultured on PDMS and TCPS based on an MPO content of 30 pmol or 2.5 μg MPO per 10 6 PMNs [42].…”

“…Depending on the surface, PMN adhesion can begin as early as 15 min after seeding and begins to decline after 2–4 h [17,39–41]. To determine if the enhanced primary granule release from PMNs on PEG hydrogels is indeed adhesion mediated, we cultured PMNs on PEG hydrogels and TCPS for 5, 15, 30, 60, 90, 120, and 240 min.…”

“…2B). MMP-9 degrades gelatin so it is likely that MMP-9 was actively cleaving the gelatin hydrogel substrate and therefore was not as freely available in the culture medium to be detected by immunoassay [17]. PMN cell viability was significantly lower on gelatin hydrogels than on PEG hydrogels or TCPS (p < 0.01) (Supplementary Fig.…”

“…Previous studies from our group showed that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG hydrogels and gelatin + PEG [GP] hydrogels) had significantly greater primary granule release than PMNs cultured on polydimethylsiloxane (PDMS) or tissue culture polystyrene (TCPS) [16,17]. To understand the underlying mechanisms of this enhanced primary granule release, we investigated the following:…”

“…We previously found that Src family kinases were involved in primary granule release from PMNs cultured on PEG hydrogels, TCPS, and GP hydrogels and in tertiary granule release from PMNs cultured on PEG hydrogels, PDMS, TCPS, and GP hydrogels [17]. PI3K-γ is a signaling molecule that has been shown to be involved in early, adhesion-independent, fMLP-induced PMN activation [24–26].…”