Anna Scislowska-Czarnecka

Enhanced early vascular permeability in gelatinase B (MMP-9)-deficient mice: putative contribution of COX-1-derived PGE2 of macrophage origin.

Increased vascular permeability leading to vascular leakage is a central feature of all inflammatory reactions and is critical for the formation of an inflammatory exudate. The leakage occurs because of gap formation between endothelial cells and breakdown of the basement membrane barriers. The present study aimed to investigate the role of gelatinase B [matrix metalloproteinase 9 (MMP‐9)], known to be involved in neutrophil exudation, in changes of vascular permeability at the early stages of acute zymosan peritonitis. We show that although MMP‐9 is being released already within the first minutes of peritonitis, its lack, induced pharmacologically or genetically, does not decrease but rather increases vasopermeability. In mice treated with an inhibitor of gelatinases (A and B), a tendency to increased vasopermeability existed, and in MMP‐9−/− mice [knockout (KO)], the difference was statistically significant in comparison with their controls. Moreover, in intact KO mice, significantly augmented production of prostaglandin E2 (PGE2) of cyclooxygenase 1 (COX‐1) origin was detected, and depletion of peritoneal macrophages, but not mast cells, decreased vasopermeability in KO mice. Thus, the increase of vasopermeability observed on KO mice is a result of the increased production of COX‐1‐derived PGE2 by peritoneal macrophages. We conclude that genetic deficiency in gelatinase B might lead to the development of a compensatory mechanism involving the COX pathway.

Effects of Aliphatic Polyesters on Activation of the Immune System: Studies on Macrophages

There is a constant search for biodegradable polymers with biocompatible characteristics. However, the reported materials are rarely tested for their immunostimulatory properties, which is an important issue as immune cells activated by the polymers might cause their rejection and lead to further injury to the host tissues. Therefore, the aim of the present study was to determine if biodegradable polymers are able to activate RAW 264.7 macrophages. Aliphatic polyesters, poly(L-lactide) (PLLA), poly(L-lactide-co-trimethylene carbonate) (PLTMC), poly(glycolide-co-L-lactide) (PGLA), poly(glycolide-co-L-lactide-co-ε-caprolactone) (PGLCap) and poly(glycolide-co-ε-caprolactone) (PGCap), processed into foils by slip-casting, were characterized in terms of their structure (1H-NMR, GPC, DSC) and surface properties (chemical composition, water contact angle, surface free energy, topography and roughness). RAW 264.7 cells were cultured on the materials for 3 or 5 days and their adherence, numbers of apoptotic/necrotic cells, as well as production of several cytokines/chemokines and other inflammation-related molecules (matrix metalloproteinases, nitric oxide) was evaluated. The study demonstrated that PLLA and PGLA did not influence macrophage activation and survival. In contrast, PLTMC, PGLCap and PGCap significantly decreased macrophage adherence, increased ratio of apoptosis and up-regulated synthesis/release of numerous inflammatory mediators. Thus, the latter materials might initiate an undesired inflammatory reaction. The above effects of the polymers were attributed to their high hydrophobicity and low polarity due to the presence of ε-caproyl blocks (PGLCap and PGCap), and/or high flexibility and susceptibility to mechanical deformation due to low glasstransition temperature (PLTMC, PGLCap and PGCap). In conclusion, while PLLA and PGLA do not affect macrophage functioning, the other materials (PLTMC, PGLCap, PGCap) up-regulate macrophage activity.

Effects of macrophage depletion on peritoneal inflammation in swiss mice, edible frogs and goldfish.

SWISS mice, edible frogs and goldfish i.p. injected with zymosan (Z groups) develop peritoneal inflammation connected with a massive intraperitoneal accumulation of leukocytes, which is significantly diminished in mice and fish (but not frogs) by supplementation of zymosan with morphine (ZM groups). In order to check the putative role of resident peritoneal macrophages in morphine-modulated zymosan-induced peritonitis, some animals were depleted of resident macrophages by repeated i.p. injections of clodronate-liposomes (CL) followed by Z or ZM injection. In SWISS mice such CL-induced removal ofMac-3-positive cells (macrophages) resulted in an enhanced influx and prolonged accumulation of polymorphonuclear leukocytes (PMNs) in CL-Z and CL-ZM groups in comparison with their counterparts with intact macrophages. Nevertheless, supplementation of zymosan with morphine inhibited the early stages of peritonitis in CL-treated animals as it did in untreated mice. This indicates that intact peritoneal macrophages of SWISS mice are important for limiting PMN accumulation, perhaps mainly through the release of IL-10, but are not critical for the induction of anti-inflammatory effects of morphine during the early stages of peritonitis. Unexpectedly, macrophage depletion in CL-treated frogs and fish resulted in a lack of a typical peritonitis in both Z and ZM groups of these ectothermic animals.

Met-enkephalin involvement in morphine-modulated peritonitis in swiss mice.

Morphine coinjection with zymosan inhibits pain and leukocyte accumulation during peritonitis in several strains of mice, and affects systems of endogenous opioids. Present investigations focus on Met-enkephalin (Met-ENK) in the inflamed peritoneal cavity and brain centers of Swiss mice. Males of Swiss mice were IP injected with zymosan or zymosan supplemented with morphine. At the selected time points the peritoneal leukocytes were counted and the Met-ENK level was measured in exudatory fluid and leukocytes, striatum, hypothalamus, and pituitary gland. The Met-ENK content in peritoneal fluid rised sharply after zymosan injection, which corresponded with its decline in exudatory leukocytes, hypothalamus, and striatum. Morphine coinjection enhanced intraperitoneal accumulation of Met-ENK and its release from exudatory leukocytes, but inhibited its early fluctuations in hypothalamus and striatum. Effects of morphine-modulated inflammation on the Met-ENK system lasted longer than 7 days.

Oxygen plasma surface modification augments poly(L‐lactide‐co‐glycolide) cytocompatibility toward osteoblasts and minimizes immune activation of macrophages

Here, we report on modification of one of the model biomedical polymers, poly L-lactide-co-glycolide (PLGA; 85:15), by reactive ion etching (RIE) oxygen plasma treatment. PLGAs major disadvantage is high hydrophobicity which restrains binding of cell-adhesive proteins and host cells. In the current approach, we aimed to answer two questions: (1) will only short (10 s) and moderate (20-200 mTorr, 45-90 W) RIE oxygen plasma treatment, leading to decrease of water contact angle by only up to 10°, sufficiently improve PLGA adherence to cells, and (2) how will this affect osteoblasts and activation of the immune system? All obtained modified PLGAs had improved hydrophilicity but unaltered roughness (as revealed by water contact angle measurements, X-ray photoelectron spectroscopy, and atomic force microscopy) resulting in significantly improved adhesion of osteoblasts (MG-63) and their low activation. Importantly, macrophages (RAW 264.7), one of the key cells initiating inflammation and bone resorption, responded significantly less vigorously to the modified polymers, expressing/releasing lower amounts of nitric oxide, matrix metalloproteinases (MMP-9), and pro-inflammatory cytokines (TNF-α, IL-6, IL-12p70, IFN-γ, IL-10). We conclude that already slight RIE oxygen plasma modification of PLGA is sufficient to improve its surface properties, and enhance cytocompatibility. Most importantly, this type of modification prevents excessive immune response.

Challenges in 3D culturing of neutrophils : assessment of cell viability

Standard cell culturing on plastic plates (two dimensional (2D) cultures) does not represent the actual microenvironment where cells reside in tissues. The three dimensional (3D) systems, composed of extracellular matrix and/or pure amino acids which form a scaffold for cells, are more accurate in this respect. 3D cultures were primarily developed for cancer cells but there is also a need for their application in studies on inflammatory leukocytes. Herein we describe our approach to study neutrophil-like cells in the 3D system. We describe measures taken to establish a neutrophil-like cell line (nHL-60) and selection of 3D scaffolds (PuraMatrix alone or enriched with collagen type I) for their culturing. We focus on challenges in measurement of neutrophil viability in 3D cultures and based on our data we suggest application of resazurin, rather than tetrazolium-based dyes or trypan blue exclusion, for evaluation of neutrophil viability.