Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT01862068 |
| Other study ID # |
NI 10017 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
May 25, 2012 |
| Est. completion date |
May 18, 2018 |
Study information
| Verified date |
April 2021 |
| Source |
Assistance Publique - Hôpitaux de Paris |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Anti-neutrophil cytoplasm antibodies (ANCA)-associated vasculitis are rare diseases
characterized by inflammation of blood vessels. Among the numerous cell types that play a
role in vasculitis, one of the key actors is the neutrophil. Neutrophils are equipped with
very powerful molecules that they use to destroy the invading microbes. Therefore, the
mechanisms controlling neutrophil activation should be tightly controlled. If that is not the
case, neutrophils may destroy the tissues of the host. This is what happens during chronic
inflammation in vasculitis. Autoantibodies directed against neutrophils, ANCA, produced thus
demonstrating that neutrophils are also targets of the immune system in these diseases. In
addition, molecular studies provided evidence that genes normally silenced in mature
neutrophils under normal conditions can be re-expressed in neutrophils from patients with
ANCA-associated vasculitis thus strongly suggesting a profound deregulation of neutrophil
functions in these conditions. Notably, the investigators have preliminary data showing that
neutrophils from patients with granulomatosis with polyangiitis (GPA, formerly Wegener's
granulomatosis), an ANCA-associated vasculitis, interfere with the normal phase of resolution
of inflammation.
The objective of the investigators' study is to understand the mechanisms underlying this
increased activation state and determine if neutrophils could be used to define prognostic
markers by clinicians to optimize patients' care. Therefore, the investigators plan to study
the expression of proteins implicated in GPA pathophysiology at the membrane of neutrophils
when they undergo apoptosis. The investigators will also study the deregulation of protein
expression in neutrophils. This point will be the molecular translation of neutrophil
deregulation. This technique is powerful and well adapted to identify by mass spectrometry
the proteins that will be differentially expressed between the control and the disease state.
After identification of proteins differentially expressed in patients with GPA, the
investigators will further investigate whether their expression is modulated during the
disease course and/or modified by the treatment.
The investigators believe that understanding these neutrophil perturbations can lead to
better monitoring of disease activity. Ultimately, the investigators may propose more
targeted anti-inflammatory therapies which would be better tolerated by patients. the
investigators also can identify new markers for disease activity which allow clinicians to
define a better therapeutic strategy.
Description:
A - Specific Aims General aim: This project is designed to study physiopathological
mechanisms involved in GPA at the cellular and molecular level. the investigators want to
study neutrophils as prognostic factors and determine the implication of PR3, the major
autoantigen in the resolution phase of inflammation.
the investigators' working hypothesis is that structural and functional analysis of PR3 in
neutrophils will help the investigators to unravel its role in GPA. Hence, our long-term
objective is to better understand the mechanisms involved in the modulation of PR3 expression
at the membrane of neutrophils in order to inhibit their activation and propose novel
anti-inflammatory therapeutic strategies adapted to GPA.
Present project: The investigators recently demonstrated that in human neutrophils i) PR3 can
be expressed at the membrane of neutrophils during apoptosis in the absence of degranulation,
ii) PR3 is a member of a molecular scaffold comprising phospholipidscramblase1 (scramblase)
and calreticulin externalized during apoptosis, iii) apoptosis-induced PR3 membrane
expression impairs macrophage reprogramming after phagocytosis of apoptotic neutrophils The
investigators' hypothesis is that PR3 might have specific molecular partners which can
facilitate its externalization and/or its persistence at the plasma membrane during
neutrophil apoptosis thus potentiating its auto-antigenic capacity.
The specific aims are:
1. Characterization of apoptosis in neutrophils from GPA patients
2. Analysis of PR3 partners (CD16, CD11b, Calreticulin, Phospholipidscramblase1) or
co-expressed protein of interest in inflammation (AnnexinA1,) expression in neutrophils
from patients with GPA compared to healthy controls
3. Identification of new partners of PR3 in neutrophils
4. Analysis of the proteome of neutrophils from GPA patients
B. Background and Significance 1.1. Introduction GPA is a systemic vasculitis which is mainly
associated with anti-PR3 ANCA. The role of activated neutrophils in vasculitis lesions is
suggested by their presence in perivascular infiltrates, especially in kidney glomeruli and
in the lung.
PR3 is a serine protease first described within azurophilic granules of neutrophils which are
NOT supposed to fuse with the plasma membrane but are released within the phagolysosome after
phagocytosis of the pathogen. We have shown for the first time that the subcellular
localization of PR3 was not restricted to azurophilic granules, as previously thought, but
that PR3 was localized in secretory vesicles, a highly mobilizable compartment, thus
explaining the presence of PR3 at the surface of resting isolated neutrophils. Moreover, a
high percentage of neutrophils expressing PR3 observed in vasculitis patients could be
considered as a risk factor for vasculitis. This high expression of membrane PR3 in patients
with GPA resulted in an increase in ANCA-induced respiratory burst in neutrophils thus
enhancing the inflammatory process. In vitro the binding of ANCA to PR3 trigger the
respiratory burst, degranulation and the release of pro-inflammatory cytokines.
Because PR3 membrane expression is an important pathophysiological event in GPA since it is a
prerequisite for the subsequent activation of neutrophils by ANCA, we sought to determine
mechanisms involved in PR3 membrane expression. In neutrophils, we demonstrated that
apoptosis triggered a significantly increased membrane PR3 expression without degranulation.
In these experiments, we failed to detect MPO at the membrane of neutrophils. Our results
differ from previously reported data showing that both MPO and PR3 were externalized during
apoptosis because of the translocation of azurophilic granules to the plasma membrane. This
phenomenon seems to occur when neutrophils undergo late apoptosis and secondary necrosis,
leading to expression of all granule proteins on the cell surface.
Apoptosis-induced PR3 externalization could be of great relevance in the pathophysiology of
GPA because PR3, which was supposed to be an intracellular antigen, is thus exposed at the
plasma membrane of apoptotic cells thus amplifying its pro-inflammatory potential, its
immunogenic properties and interfering with macrophage phagocytosis. We have also
demonstrated that during apoptosis PR3 expression is associated with the expression of
calreticulin, a well-known "eat-me signal". We have finally evidenced that the presence of
PR3 impairs the reprogramming of macrophage to an anti-inflammatory profile after
phagocytosis of apoptotic cells through the CRT/LRP pathway.
It can be hypothesized that the expression of the complex PR3/scramblase/CRT and other
partners (CD11b, CD16) at the surface of apoptotic neutrophils could constitute good
prognosis markers.. Gene array studies have pointed out a deregulation of neutrophils in GPA.
We make the hypothesis that the proteome of neutrophils of GPA patients would be very
informative of its activation state, survival state and that it could be a biological marker
of clinical severity.
It is thus pertinent to study the deregulation of neutrophils during their all life cycle:
their oxidative burst under activation, their physiologic apoptosis, the expression of PR3
partners. This study will be performed in neutrophils from patients with GPA compared to age
and sex adjusted healthy controls.
C. Preliminary studies Analysis of apoptosis in neutrophils from GPA patients In the
literature, neutrophils from patients with ANCA associated vasculitis (AAV) were found to
have a disturbed TNFα-induced apoptosis and tends to have a higher rate of apoptosis than
controls. However, published data on physiologic apoptosis which is the spontaneous apoptosis
that neutrophils undergo in the absence of exogenous stimuli is contradictory. When calf
foetal serum is added in medium, neutrophils from GPA patients display a similar apoptosis
rate than controls. When the serum of patients is used, neutrophils from AAV patients in
remission have a higher survival rate in vivo than controls, and this difference disappear
when normal serum is used. These results are in agreement with the discovery that GPA
patients have a high expression of PCNA which is associated with survival. Therefore, we want
to study the apoptosis rate of neutrophils in GPA at diagnosis, during relapse and after
remission on whole blood. These results will be compared to age and sex matched controls.
Apoptosis will be evaluated by the phosphatidylserine externalization measured by flow
cytometry after annexin-V labelling and 7-AAD staining on CD15+ cells. PCNA expression will
be also investigated at the different clinical stage and proteomic profile of apoptotic
neutrophils will be performed.
Analysis of scramblase/ Calreticulin expression in neutrophils from GPA patients In a
cellular model (RBL cell line), we have demonstrated that scramblase extinction by shRNA was
associated with a decreased expression of phosphatidylserine during apoptosis and that PR3
apoptosis induced expression was also impaired (unpublished data). We were also able to
demonstrate that in vitro scramblase and PR3 physically interact. During apoptosis, PR3 is
also associated with calreticulin at the membrane of neutrophils. These data suggest that the
association PR3/Scramblase/Calreticulin form a molecular scaffold which is a functional
platform which could interfere with recognition pathways (like CRT/LRP) between macrophages
and apoptotic neutrophils. The expression of these partners at the neutrophil membrane during
apoptosis will be investigated in patients at diagnosis and when remission is obtained.
Research design and Methods:
1. Characterization of apoptosis in neutrophils from GPA patients We will determine
apoptosis rate of neutrophils in whole blood.
Methodology:
Research will be performed both on neutrophils isolated from healthy control donors
obtained in the French Blood Institute (Etablissement Français du Sang) and from
vasculitis patients from the Department of Internal Medicine of Cochin Hospital headed
by Professor Loïc Guillevin.
Apoptosis will be induced "physiologicaly" in whole blood by overnight incubation at
37°C.
Phosphatidylserine externalization will be measured by flow cytometry after annexin-V
labeling associated with 7-AAD staining for CD15+ cells.
Western blot analysis using a specific antibody against PCNA will also be used to
visualize its expression.
2. Analysis of scramblase, PR3, calreticulin, AnnexinA1, expression in apoptotic
neutrophils from GPA patients We will determine the membrane expression of PR3, CRT,
PLSCR, AxA1, in apoptotic neutrophils in the different subsets of neutrophils: non
apoptotic ones (AnnexinV-, 7AAD-), early apoptotic neutrophils (AnnexinV+, 7AAD-) and
late apoptotic neutrophils (AnnexinV+, 7AAD+). This determination will be done for
patients at the time of diagnosis (in active state; BVAS>3) and at remission.
Methodology
- Neutrophils will be isolated from blood using Dextran gradient and Ficoll.
- Investigation of PR3, CRT, PLSCR, AxA1, membrane expression will be measured on
isolated neutrophils by flow cytometry after overnight incubation at 37°C with
assessment of apoptosis by annexin-V labeling and 7AAD staining.
3. Study of the respiratory burst in neutrophils of GPA patients One of the main functions
of neutrophils is the production of reactive oxygen species, we will determine the
oxidative metabolism of neutrophils due to different stimuli (f-MLP, opsonized zymosan
beads by complement, opsonized zymosan beads by IgG, PMA) with or without a previous
priming by TNFα.
Methodology:
Exploration of the respiratory burst will be done in whole blood with a chemoluminescent
(luminol) technique. Neutrophils will be primed by TNFα or stimulated without priming.
The burst response will be analysed during 90 minutes after stimulation.
4. Deregulation of neutrophil Cytokinome Neutrophils are capable to synthetize a large
number of cytokines (IL-1, TNFα, IL-6, IL-12), growth factors like GM-CSF and
chimiokines like IL-8. More recently it has been shown that neutrophils are also great
producers of BAFF (B-cell activating factor) and that production is superior to those by
monocytes or dendritic cells when stimulated by INF-γ. In GPA serum BAFF levels have
been shown to be higher than controls and recently targeting B-lymphocytes therapeutics
have confirmed in large controlled trials their importance in AAV.
Methodology:
Serum of patients will be collected at the same time than neutrophils. They will be
analyzed using BD™ Cytometric Bead Array (CBA) with detection of IL-8, IL-1β, IL-6,
IL-10, TNF, and IL-12p70. Neutrophils which will be isolated as in 1) will be stimulated
by INF-γ, LPS and G-CSF to analyze their production and secretion of BAFF/BlyS
determined by ELISA.
5. Deregulation of neutrophil Proteome and functional study of new PR3 partners Gene array
studies have shown a probable deregulation of neutrophils in AAV, in this study we will
study this deregulation at the protein level.
Methodology:
To study the proteome of GPA patient when active and in remission compared to healthy
controls we will use a differential in gel electrophoresis of cytosolic samples of
neutrophils obtained by sonication. Identification of proteins of interest will be done by
mass spectrometry MALDI-TOF-TOF. Functional validation of these proteins by over-expression
or siRNA extinction will be performed.
