Peripheral blood natural killer cell percentages in granulomatosis with polyangiitis correlate with disease inactivity and stage

Abstract

IntroductionThe role of CD3−CD56+ natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. Recently, it has been shown that peripheral blood NK cells can kill renal microvascular endothelial cells, suggesting a pathogenic role of NK cells in this disease. So far, subset distribution, phenotype, and function of peripheral blood NK cells in relation to GPA disease activity have not been elucidated. Moreover, it is not known whether NK cells infiltrate GPA tissue lesions.MethodsParaffin sections of GPA granulomas and controls were stained with anti-CD56 and anti-CD3 antibodies. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. NK cell degranulation was analyzed using cocultures of patient PBMCs with target cells and surface expression of CD107a. Clinical data were extracted from medical records. Statistical analysis was performed in an exploratory way.ResultsCD56+ cells were not detectable in active granulomatous GPA lesions but were found frequently in granulomas from tuberculosis and sarcoidosis patients. In GPA, the proportion of NK cells among peripheral blood lymphocytes correlated negatively with the Birmingham Vasculitis Activity Score (BVAS) (n = 28). Accordingly, NK cell percentages correlated positively with the duration of remission (n = 28) and were significantly higher in inactive GPA (BVAS = 0, n = 17) than in active GPA, healthy controls (n = 29), and inactive control diseases (n = 12). The highest NK cell percentages were found in patients with long-term remission and tapered immunosuppressive therapy. NK cell percentages >18.5 % of peripheral blood lymphocytes (n = 12/28) determined GPA inactivity with a specificity of 100 %. The differentiation into CD56dim and CD56bright NK cell subsets was unchanged in GPA (n = 28), irrespective of disease activity. Similar surface expression of the activating NK cell-receptors (NKp30, NKp46, and NKG2D) was determined. Like in healthy controls, GPA NK cells degranulated in the presence of NK cell receptor ligand bearing epithelial and lymphatic target cells.ConclusionsNK cells were not detectable in GPA granulomas. Peripheral blood NK cell percentages positively correlate with the suppression of GPA activity and could serve as a biomarker for GPA activity. Peripheral blood NK cells in GPA patients are mature NK cells with preserved immune recognition.

Multicolor flow cytometric analysis of TLR2 and TLR9 expression and function in NK cells from patients with ANCA‐associated vasculitis

“…Interestingly, we did not find any differences in the number and proportion of PB NK cells in patients with AAV relative to HC (except for the proportion of CD56 dim NK cells, which were lower in ANCA-negative patients with respect to HC) and, accordingly, only rare NK cells were detected in the renal biopsy taken from a single patient. This finding is in line with similar observations in patients with GPA granulomas (32). Hence, there seems to be a negligible contribution of NK cells to local inflammatory responses in AAV.…”

“…NK cell activation through TLR9 is accessory cytokine-dependent, hence the addition of low (suboptimal) IL-12 doses was needed to induce TLR9 ligand stimulation. Similarly to another study of patients with GPA, we did not observe any significant differences in NK cell degranulation measured as CD107a expression between AAV patients and HC following stimulation with K562 target cells (32). A similar trend towards increased IFN-g production in AAV patients was found after TLR2 ligand stimulation.…”

“…Despite bacterial contribution in AAV pathogenesis hypothesized in this paper, other possibilities not yet explored remain open. Similarly to another study of patients with GPA, we did not observe any significant differences in NK cell degranulation measured as CD107a expression between AAV patients and HC following stimulation with K562 target cells (32). It is plausible that NK cells require additional stimuli for degranulation as an in vitro study of GPA patients supports; the authors reported increased CD107a degranulation in NK cells in the presence of either PR-3 or TNF-stimulated renal microvascular endothelial cells in patients relative to HC (31).…”

“…As observed with in vitro experiments, decreased NK cell numbers in autoimmune disease could be a consequence of cytokine-induced apoptosis serving to stimulate NK cells (33); however, preferential recruitment by affected tissues cannot be ruled out, as observed in patients with type 1 diabetes mellitus, psoriasis, and rheumatoid arthritis (34)(35)(36). This finding is in line with similar observations in patients with GPA granulomas (32). This finding is in line with similar observations in patients with GPA granulomas (32).…”

“…Although many studies report decreased NK cell numbers or impaired NK cell cytotoxicity in the PB of patients with autoimmune diseases (27)(28)(29)(30), others performed in AAV patients produced inconsistent results: one reports reduced NK cell numbers with respect to HC (29) and the others report comparable proportions of NK cells (18,31,32). As observed with in vitro experiments, decreased NK cell numbers in autoimmune disease could be a consequence of cytokine-induced apoptosis serving to stimulate NK cells (33); however, preferential recruitment by affected tissues cannot be ruled out, as observed in patients with type 1 diabetes mellitus, psoriasis, and rheumatoid arthritis (34)(35)(36).…”