Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMNs). Here, we describe the development of a subgroup of human PMNs expressing CCR5, termed CCR5+ PMNs. Auto- and paracrine tumor necrosis factor (TNF) signaling increases intracellular neutrophil elastase (ELANE) abundance and induces neutrophil extracellular traps formation (NETosis) in CCR5+ PMNs, and triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, known activators of NETosis. In vivo, we find an increased number of CCR5+ PMNs in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ PMNs. In conclusion, we identify a phenotype of pro-NETotic, CCR5+ PMNs present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value. Display omitted •Priming induces neutrophil diversification into CCR5− and CCR5+ cells•TNFR2 signaling renders CCR5+ neutrophils pro-NETotic by ELANE upregulation•CCR5+ neutrophils appear in the lamina propria of ulcerative colitis patients•Retrograde TNF signaling induces NETosis of CCR5+ neutrophils Neuenfeldt et al. show that neutrophil priming induces dichotomous expression of CCR5. TNFR2 signaling increases ELANE expression in CCR5+ neutrophils. CCR5 triggering or reverse signaling of membrane-bound TNF activates ELANE, leading to enhanced NETosis. Pro-NETotic CCR5+ neutrophils in the lamina propria of ulcerative colitis patients may affect the success of anti-TNF therapies.