The neutrophil subset defined by CD177 expression is preferentially recruited to gingival crevicular fluid in periodontitis.

In recent years, the concept of distinct subpopulations of human neutrophils has attracted much attention. One bona fide subset marker, exclusively expressed by a proportion of circulating neutrophils in a given individual, and therefore dividing neutrophils in two distinct subpopulations, is the glycoprotein CD177. CD177 is expressed on the plasma and granule membranes of 0-100% of circulating neutrophils depending on the donor. Several in vitro studies have linked CD177 to neutrophil transmigration, yet very few have looked at the role of CD177 for tissue recruitment in vivo. We investigate whether the CD177+ and CD177- neutrophil subsets differ in their propensity to migrate to both aseptic- and microbe-triggered inflamed human tissues. Microbe-triggered neutrophil migration was evaluated in samples of gingival crevicular fluid (GCF) from patients with periodontitis, whereas neutrophil migration to aseptic inflammation was evaluated in synovial fluid from patients with inflammatory arthritis, as well as in exudate from experimental skin chambers applied on healthy donors. We found that the proportion of CD177+ neutrophils was significantly higher in GCF from patients with periodontitis, as compared to blood from the same individuals. Such accumulation of CD177+ neutrophils was not seen in the two models of aseptic inflammation. Moreover, the proportion of CD177+ neutrophils in circulation was significantly higher in the periodontitis patient group, as compared to healthy donors. Our data indicate that the CD177+ neutrophil subset is preferentially recruited to the gingival crevice of periodontitis patients, and may imply that this subtype is of particular importance for situations of microbe-driven inflammation.

In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals.

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.

Neutrophil recruitment to inflamed joints can occur without cellular priming.

Recruitment of neutrophils from blood to tissues is a cardinal event in inflammation during which neutrophils switch from a resting, naive state to a preactivated, primed phenotype; the priming process is characterized by alterations in the composition of cell surface adhesins, for example, shedding of l-selectin and mobilization of granule-stored integrins to the cell surface. Ligation of chemotactic receptors and interactions with the endothelial lining are established triggers of neutrophil priming and in line with this, in vivo transmigrated neutrophils obtained from tissues are typically highly primed. We here characterize the priming of neutrophils brought about by in vivo recruitment from blood to inflamed joints by the analyses of synovial fluid and blood from patients with inflammatory arthritis. For comparisons, we used controlled in vivo models of neutrophil transmigration to skin of healthy subjects. In contrast to the residing view and in vivo transmigrated neutrophils from skin models, neutrophils from synovial fluid were often surprisingly resting and phenotypically very similar to naive cells isolated from peripheral blood; synovial fluid cells often retained l-selectin and had undergone minimal up-regulation of integrin receptors. In complete agreement with our in vivo findings, cell-free synovial fluid was potently chemotactic without triggering alteration of surface receptors also in vitro. We conclude that tissue recruitment of neutrophils does not by default trigger l-selectin shedding and granule mobilization, and the chemoattractant(s) guiding neutrophils to synovial fluid apparently operate without inducing cellular priming.

Collection of in vivo transmigrated neutrophils from human skin.

A wealth of knowledge on the life and death of human neutrophils has been obtained by the in vitro study of isolated cells derived from peripheral blood. However, neutrophils are of main importance, physiologically as well as pathologically, after they have left circulation and transmigrated to extravascular tissues. The journey from blood to tissue is complex and eventful, and tissue neutrophils are in many aspects distinct from the cells left in circulation. Here we describe how to obtain human tissue neutrophils in a controlled experimental setting from aseptic skin lesions created by the application of negative pressure. One protocol enables the direct analysis of the blister content, infiltrating leukocytes as well as exudate fluid, and is a simple method to follow multiple parameters of aseptic inflammation in vivo. Also described is the skin chamber technique, a method based on denuded skin blisters which are subsequently covered by collection chambers filled with autologous serum. Although slightly more artificial as compared to analysis of the blister content directly, the cellular yield of this skin chamber method is sufficient to perform a large number of functional analyses of in vivo transmigrated cells.

A simple skin blister technique for the study of in vivo transmigration of human leukocytes.

The study of human leukocytes is almost exclusively conducted using cells isolated from peripheral blood. This is especially true for neutrophils, despite the fact that these cells are of main (pathological) importance in extravascular tissues upon e.g., infection and/or tissue damage. The journey from circulation to tissue is typically associated with a number of cellular changes, making the cells primed, or hyper-responsive, and in many aspects distinct from the cells present in circulation. Models to obtain in vivo transmigrated leukocytes from human tissue are available, but not widely used. We describe here an easy-to-use model for the study of local inflammation, stemming from limited tissue damage, which can be used to isolate viable and functional leukocytes. The model is based on the generation of aseptic skin blisters, formed by the application of negative pressure, and allows for investigations of the cellular infiltrate as well as of soluble mediators present in the exudate. We believe that this method, combined with modern analysis equipment suitable for small volumes and cell numbers, could be of great use for increasing our understanding of the nature and function of leukocytes that have left circulation and transmigrated to inflamed tissues.