Neutrophil surface presentation of the anti-neutrophil cytoplasmic antibody-antigen proteinase 3 depends on N-terminal processing

Abstract

The neutrophil serine protease proteinase 3 (PR3) is a main autoantigen in anti-neutrophil cytoplasmic antibody-associated vasculitis. PR3 surface presentation on neutrophilic granulocytes, the main effector cells, is pathogenically important. PR3 is presented by the NB1 (CD177) glycoprotein, but how the presentation develops during neutrophil differentiation is not known. An N-terminally unprocessed PR3 (proPR3) is produced early during neutrophil development and promotes myeloid cell differentiation. We therefore investigated if PR3 presentation depended on NB1 during neutrophil differentiation and if PR3 and proPR3 could both be presented by NB1. In contrast to mature neutrophils, differentiating neutrophils showed an early NB1-independent PR3 surface display that was recognized by only two of four monoclonal anti-PR3 antibodies and occurred in parallel with proPR3, but not PR3 secretion, suggesting that the NB1-independent surface PR3 was proPR3. PR3 gene expression preceeded NB1. When the NB1 receptor was detected on the surface, a mode of PR3 surface display similar to mature neutrophils developed together with the degranulation system. Ectopic expression studies showed that NB1 was a sufficient receptor for PR3 but not proPR3. ProPR3 display on the plasma membrane may influence the bone marrow microenvironment. NB1-mediated PR3 presentation depended on PR3 N-terminal processing implicating the PR3-N-terminus as NB1-binding site.

Fig. 1

NB1 and proteinase 3 (PR3) surface display during neutrophilic differentiation in vitro. (a) Typical example of NB1 and PR3 surface presentation on live cells using 12·8, 4A5, 6A6 and MCPR3-2 monoclonal anti-PR3 antibodies that give two different patterns of membrane PR3 development. Confocal microscopy (b) demonstrates that the MCPR3-3 anti-PR3 antibody recognizes surface PR3 on developing neutrophils without NB1. (c) Quantification of NB1 and PR3 surface display development. Percentages of membrane-positive cells for either NB1 or PR3 cells are shown with the isotype set to 5% positive cells; n = 4 independent experiments per time-point during 10 days of differentiation.

Fig. 2

NB1 and proteinase 3 (PR3) surface display on neutrophils from peripheral blood. The correlation coefficient between percentage of PR3 and NB1-positive neutrophils was R = 0·99 in all cases, independent of the antibodies used (adult neutrophils [polymorphoneutrophil (PMN)]: n = 26 for 12·8, n = 22 for 4A5, n = 8 for MCPR3-2, n = 6 for 6A6). Neonatal, cord blood neutrophils (CB-PMN): n = 34 for 12·8, n = 8 for 4A5, n = 4 for MCPR3-2, n = 5 for 6A6).

Fig. 3

Proteinase 3 (PR3) and NB1 gene expression during neutrophil differentiation. (a) PR3-mRNA showed a gradual decline during in vitro differentiation, NB1 mRNA was up-regulated after addition of granulocyte–colony-stimulating factor and remained elevated during the observation period. Values are shown as percentage of housekeeper (18s). One typical of four independent experiments is shown. (b) PR3 and NB1 subcellular distribution in granules of peripheral blood neutrophils. Subcellular distribution is an indicator for the time of expression during development. PR3 was stored mainly in primary granules (OD = optical density: primary granules (1°): 94 ± 16, secondary/tertiary granules (2°/3°): 16 ± 4, secretory vesicles and plasma membranes (m./s.v) 11 ± 2, cytosol (c) 13 ± 2), NB1 in secondary and tertiary granules (OD: primary granules: 23 ± 15, secondary/tertiary granules: 100 ± 0, secretory vesicles and plasma membranes 28 ± 6, cytosol 14 ± 2). Immunoblot of a typical experiment and densitometric analysis from three (NB1) or four (PR3) independent experiments are shown.

Fig. 4

Proteinase 3 (PR3) and NB1 protein expression during neutrophilic differentiation. PR3 as detected by immunoblot was processed to a 29 kDa form by day 2 in the cells; in the cell culture supernatant, a 35 kDa proPR3 appeared on day 1; the 29 kDa PR3 was only detected from day 7. NB1 appeared in the cells on day 2 and in the supernatant on day 7. Lane loading was normalized for cell number and cell concentration respectively. PR3 was assessed by MCPR3-2 and polyclonal rabbit anti-PR3 giving similar results. A typical of three independent differentiations is shown.

Fig. 5

Proteinase 3 (PR3) binding to neutrophil progenitors and translocation to the plasma membrane. (a) Binding of purified neutrophil PR3 to neutrophilic progenitors (days 3–6 of differentiation) was assessed by flow cytometry. Percentages of membrane-positive cells for PR3 and NB1 from six independent experiments are shown (*P < 0·05). (b) Degranulation in response to 20 min stimulation with 10⁻⁶ M N-formyl-Met-Leu-Phe (fMLP) as percentage increase in membrane display of surface anchored marker molecules for primary granules (CD66b), secondary/tertiary (CD11b) and secretory vesicles (CD35) (MFI = mean fluorescence intensity). The increase in translocation after differentiation was especially marked for mature PR3 (*P < 0·05). Means of three independent differentiations are shown.

Fig. 6

Surface presentation of NB1, pro proteinase 3 (proPR3) and PR3 expressed in HEK293 cells. Surface PR3 is exclusively up-regulated after co-transfection with NB1. There was no significant surface expression in single transfection of either proPR3 or PR3. Anti-PR3 12·8 was used in these experiments; similar data were obtained with MCPR3-2 and 4A5 (n = 5 independent transfections, *P < 0·05).