Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. In vitro, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites in vivo, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon in vivo transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue in vivo. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.

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 CD177neutrophil 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 microbedriven inflammation.

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.

Atypical c‐ANCA staining patterns with reactivity to some neutrophils can be explained by auto‐antibodies directed to subtype marker OLFM4.

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BackgroundAlemtuzumab (ALZ) is an immune reconstitution therapy for treating relapsing-remitting multiple sclerosis (RRMS). However, ALZ increases the risk of secondary autoimmune diseases (SADs).ObjectiveWe explored whether the detection of autoimmune antibodies (auto-Abs) could predict the development of SADs.MethodsWe included all patients with RRMS in Sweden who initiated ALZ treatment (n = 124, 74 female subjects) from 2009 to 2019. The presence of auto-Abs was determined in plasma samples obtained at the baseline and at 6, 12, and 24 months of follow-up, as well as in a subgroup of patients (n = 51), it was determined in plasma samples obtained at the remaining 3-month intervals up to 24 months. Monthly blood tests, urine tests, and the assessment of clinical symptoms were performed for monitoring safety including that of SADs.ResultsAutoimmune thyroid disease (AITD) developed in 40% of patients, within a median follow-up of 4.5 years. Thyroid auto-Abs were detected in 62% of patients with AITD. The presence of thyrotropin receptor antibodies (TRAbs) at the baseline increased the risk of AITD by 50%. At 24 months, thyroid auto-Abs were detected in 27 patients, and 93% (25/27) developed AITD. Among patients without thyroid auto-Abs, only 30% (15/51) developed AITD (p < 0.0001). In the subgroup of patients (n = 51) with more frequent sampling for auto-Abs, 27 patients developed ALZ-induced AITD, and 19 of them had detectable thyroid auto-Abs prior to the AITD onset, with a median interval of 216 days. Eight patients (6.5%) developed non-thyroid SAD, and none had detectable non-thyroid auto-Abs.ConclusionWe conclude that monitoring thyroid auto-Abs, essentially TRAbs, may improve the surveillance of AITD associated with ALZ treatment. The risk for non-thyroid SADs was low, and monitoring non-thyroid auto-Abs did not seem to provide any additional information for predicting non-thyroid SADs.