Table 1.
Patient characteristics.
Figure 1.
Gating strategies to distinguish between leukocyte populations.
FSc-SSc profile was used to distinguish lymphocytes, monocytes and granulocytes (A). Within the lymphocyte population, B lymphocytes, T lymphocytes, and NK cells were identified using membrane markers CD19, CD3 and CD16/CD56, respectively (B, D). Monocytes were either CD14+/CD16low or CD14low/CD16+ (C).
Table 2.
Toll-like receptor expression by peripheral blood leukocytes.
Figure 2.
Expression of mTLR2, mTLR4, and mTLR9 by Natural killer (NK) cells.
Cells in whole blood were labeled with antibodies against CD56, CD16, TLR2, TLR4, and TLR9. NK cells were gated according to light scatter profile and the expression of CD56/CD16. Proportions of mTLR2, mTLR4, and mTLR9 positive NK cells in HC and AAV patients are shown. Within patients, closed circles depict TLR expression in active patients (n = 6). Proportions of mTLR2+, mTLR4+, and mTLR9+ NK cells were significantly increased in AAV patients (p = 0.005, p = 0.03, and p = 0.05, respectively). Proportions of mTLR expressing NK cells did not differ between patients with active disease or in remission.
Figure 3.
Expression of mTLR2 and mTLR4 by monocytes.
Cells in whole blood were stained with antibodies against CD14, CD16, TLR2, and TLR4. Monocytes were gated according to light scatter profile and the expression of CD14/CD16. Representative histogram plots of mTLR2 and mTLR4 expression are shown in figures C and F, respectively. The proportions of mTLR2+ (A) and mTLR4+ (D) monocytes did not differ between AAV patients and HC. However, 7 AAV patients had an increased proportion of mTLR4+ monocytes, compared to HC (dotted line: cut-off mTLR4+ cells, calculated from HC). Expression levels per cell are shown in fig B (mTLR2), and E (mTLR4). mTLR2 levels were higher on AAV patient monocytes, compared to HC (p = 0.03), whereas mTLR4 levels did not differ significantly when comparing groups. In line with increased proportions of mTLR4+ monocytes, in part of the patients increased levels of mTLR4 were observed (dotted line: cut-off mTLR4 ΔMFI, calculated from HC) Within AAV patients, active patients (n = 6) are depicted with closed circles. Expression of mTLR2 and mTLR4 did not differ significantly between patients with active or quiescent disease.
Figure 4.
Toll-like receptor expression by monocytes from AAV patients who were nasal carriers of S. aureus (n = 13, white bars) and non-carriers (n = 25, gray bars).
Both the proportions of mTLR+ monocytes (left y-axis), as well as the level of mTLR expression (right y-axis) did not differ significantly between monocytes from nasal carriers and non-carriers. Intracellular levels of TLR9 (iTLR9) were increased in monocytes from patients who were nasal carriers of S. aureus.
Figure 5.
Expression of the activation marker CD25 by leukocyte subsets was analyzed after stimulation with TLR ligands.
Diluted whole blood was left unstimulated or treated with Lipoteichoic acid (TLR2 ligand), Lipopolysaccharide (TLR4 ligand), or CpG-ODN (TLR9 ligand). After 24 h, expression of CD25 by different leukocyte subsets was analyzed by flowcytometry (A). Leukocyte subsets were distinguished according to light scatter profile (lymphocytes, monocytes, granulocytes) and the expression of CD19 (B lymphocytes). In both AAV patients (open circles) and HC (filled circles), increased CD25 expression was observed by monocytes and granulocytes, in response to TLR2 and TLR4 stimulation. Particularly B lymphocytes were stimulated by CpG-ODN. Significantly increased CD25 expression is depicted with * (HC) and # (AAV patients).
Figure 6.
Release of cytokines after TLR stimulation in whole blood cultures was analyzed by ELISA.
Diluted whole blood was left unstimulated or treated with Lipoteichoic acid (TLR2 ligand), Lipopolysaccharide (TLR4 ligand), or CpG-ODN (TLR9 ligand). Comparable total leukocyte numbers were cultured from both AAV patients and HC (data not shown). In patient cultures, spontaneous release of TNF-α was increased, but TNF-α release upon TLR4 triggering was decreased compared to HC. A trend towards increased release of IL-10 was observed in patient cultures upon TLR2 and TLR4 triggering (p = 0.09 and p = 0.06, respectively), although this did not reach statistical significance. Release of IL-6 and IL-8 was comparable in patient and HC cultures.