Role Of Innate Immunity In The Pathogenesis Of ANCA-Associated Vasculitis

Angelica Gattamelata¹, Giovanna Peruzzi², Rossana Scrivo³, Roberta Priori³, Stefania Morrone⁴, Angela Santoni² and Guido Valesini³, ¹Sapienza University of Rome, Rome, Italy, ²Department of Molecular Medicine, Sapienza University, Rome, Italy, ³Department of Internal Medicine and Medical Specialties, Sapienza University, Rome, Italy, ⁴Department of Experimental Medicine, Sapienza University, Rome, Italy

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: ANCA, natural killer (NK) cells, toll-like receptors and vasculitis

Session Information

Title: Vasculitis I

Session Type: Abstract Submissions (ACR)

Background/Purpose:

Natural killer cells (NK) represent one of the main effectors of the innate immune response through the defense against viral infections and the production of immunoregulatory cytokines. Recent evidence shows an increase in the expression of TLR2 and TLR9 on NK cells of patients with ANCA-associated vasculitis (AAV) compared with healthy controls (HC). This is of great interest considering that infections are implicated in the pathogenesis of these diseases.
The aim of our study was to characterize NK cells in patients with AAV, assessing their number, phenotype and functional status.

Methods:

We enrolled 19 patients with AAV [M/F: 7/12; mean age 60.2 years (range 41-75)] according to the Chapel Hill criteria (11: granulomatosis with polyangiitis, 7: eosinophilic granulomatosis with polyangiitis, 1: microscopic polyangiitis) and 12 HC matched for age and sex with the patients. After obtaining informed consent, clinical data were collected and blood drawing was performed. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Lymphoprep gradient centrifugation. NK cells (CD3-CD56+) were evaluated by multi-parameter flow cytometry, distinguishing between cytotoxic CD56^dim and immunoregulatory CD56^bright NK cells, also divided into two recently identified subpopulations: memory NK cells (CD3-CD56+CD57+) and NK-22 cells (CD3-CD56+NKp44+CCR6+). In addition, the expression of TLR2 and TLR9 was evaluated on these cells. Finally, NK cells were isolated by magnetic separation to assess the state of activation through the expression of intracellular IFNg following in vitro incubation with TLR2 (Pam3CSK4) and TLR9 (CpG ODN) ligands.

Results:

We found no differences in the percentage of NK cells between patients (11.3%) and HC (12%). The percentage of CD56^dim and CD56^bright (80.5% vs 92% and 7.5% vs 8%, respectively), and the percentage of memory NK cells and NK-22 cells (0.550% vs 0.507% and 66% vs 67%, respectively) was comparable between AAV patients and HC. In NK cells, the expression of TLR2 was significantly higher in HC (14.8%) compared to patients (8.12%; p=0.0328) and TLR9 expression showed a tendency to be higher in patients (26%) compared to HC (15%), but the difference was not statistical significant. In the memory NK cells subset the expression of TLR2 was significantly decreased in patients (10.2%) compared to controls (19.2%; p=0.0434), while there was no difference in the expression of TLR9. Interestingly, the stimulation with TLR9 ligand induced a significant production of IFNg in patients (11%) compared to HC (2.8%; p=0.0071). No correlation with clinical and laboratory parameters was found.

Conclusion:

The increased production of IFNg after stimulation with the ligand of TLR9 demonstrates a state of NK activation in AAV patients. Hence, TLR9 may be involved in the pathogenesis of these diseases, strengthening the possibility that infections may promote their onset and/or exacerbations. Further experiments are needed to confirm these data and to clarify the meaning of the reduced expression of TLR2 on CD57+ NK cells of patients compared to HC.

Disclosure:

A. Gattamelata,
None;

G. Peruzzi,
None;

R. Scrivo,
None;

R. Priori,
None;

S. Morrone,
None;

A. Santoni,
None;

G. Valesini,
None.

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