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 Summary of the PhD thesis, which obtained the GREMI prize in 2006:

 

ROLE OF PROTEINASE 3 IN APOPTOSIS AND IN THE PRO-INFLAMMATORY ACTIVITY OF NEUTROPHIL: COMPARTIMENTALISED ACTIVATION OF PROCASPASE-3, IN ABSENCE OF APOPTOSIS

 

Magali Pederzoli-Ribeil

 

This work has been done in the INSERM Unit 845 (ex-507), in Necker Hospital, Paris

with the supervision of Dr. Véronique Witko-Sarsat

 

Neutrophils are the main cell in the innate immune system, involved in host defence mechanisms against pathogens. At each successive step of differentiation, they acquire different granule types: azurophilic granules, specific granules, gelatinase granules and secretory vesicles. According to dogma, proteins expressed in a granule are synthesised only at a certain stage of differentiation: this is known as the theory of "targeting by timing". Neutrophil differentiation is characterised by a strict compartmentalisation, each type of granule containing specific proteins with specialised effector functions. During their activation, neutrophils successively mobilise, in a precise order, their different granules specialised for a particular neutrophils response. Surprisingly, proteinase 3 (PR3), which belongs to the neutrophil serine proteinase homologue family, does not follow this dogma, since it is both predominantly expressed in the azurophilic granules, in secretory vesicles and at the neutrophil cell surface . Regarding to the patho-physiological pattern, PR3 is the target of the so called ANCA (« Anti Neutrophil Cytoplasmic Antibody ») auto-antibodies in a systemic vasculitis, Wegener’s granulomatosis . Importantly, a high percentage of neutrophils expressing membrane PR3 is a risk factor for inflammatory diseases , more specifically in vasculitis and rheumatoid arthritis.

 

Since PR3 does not follow the neutrophil compartmentalisation dogma, my PhD project focussed on structural and functional PR3 characterisation , specifically aiming to identify its new target . Through the study of the procaspase-3 cleavage, we would like to compare PR3 with elastase. Elastase is a serine proteinase homologue of PR3, strictly localised in azurophilic granules, which does not share the extragranular localisation of PR3 in neutrophils.

 

This work could not be performed in mature neutrophils in their last stage of differentiation, because of their short life-span, and low rate of protein synthesis. Moreover, as PR3 and elastase share very similar substrate specificity, it was impossible specifically to inhibit either PR3 or elastase and no proteinase-specific substrate was available at the start this project. Therefore, in our laboratory, we strategically chose to express stably PR3 or elastase in a specific cellular model, which does not endogenously express either of these two proteinases. Our model however, did contain all the enzymatic equipment required for the synthesis, maturation, targeting and storage of these proteinases. We transfected human (HMC1) and rat (RBL) mast cell lines, with PR3 and elastase cDNA. We also established a cell line over-expressing a mutated form of PR3 where the serine of the catalytic site was replaced by alanine, thus inactivating the proteinase activity. These cells over-expressing PR3 appeared to be a good model system to study the structural and functional PR3 properties and could be considered as a relevant neutrophil model.

The first part of my work studied PR3 and elastase localisation in stably transfected cells (RBL) over-expressing PR3 or elastase. We demonstrated the presence of an extra-granular pool of PR3, mobilised during apoptosis, at the cell surface, in the absence of degranulation. Moreover, PR3 externalisation was linked to phosphatidylserine externalisation. However, in the same experimental conditions, elastase was not externalised, concomitantly with phosphatidylserine (1). We therefore studied PR3 target associated proteins involved in the plasma membrane flip-flop process, that occurs during apoptosis. We have described phospholipid scramblase 1 (PLSCR1) as a PR3 membrane partner, which takes part in its externalisation at the apoptotic cell surface. We have also demonstrated that this externalised PR3 interferes with mechanisms of “eat-me” signal recognition and macrophage-dependent apoptotic cells phagocytosis (2).

 

The second part of my work focused on apoptosis, a key phenomenon of the resolution of inflammation, in our model of mast cell line transfectants (RBL and HMC1). We have measured a high constitutive caspase-3 activity in cells transfected with PR3, due to the direct procaspase-3 cleavage by PR3. This procaspase-3 activation and cleavage resulted in a 22 kDa active fragment in the membrane compartment (different from the 17 kDa apoptotic fragment) and was apoptosis independent. The 22 kDa caspase-3 fragment was found in mature neutrophils in which it appeared to be linked to cell survival since it disappeared after apoptosis induction. After cell treatment with pefabloc, a serine protease inhibitor, the 22 kDa fragment disappeared. Moreover we have shown, after neutrophil permeabilisation with streptolysin-O, an extragranular co-localisation between PR3 and caspase-3 (3). Overall, these results suggest the involvement of a serine protease in procaspase-3 cleavage in neutrophils. PR3 seems to be the best candidate according to our data in the transfectants. Consequently, procaspase-3 is a new membrane target of PR3 and not of elastase. Surprisingly, we have demonstrated this cleavage resulting in the 22 kDa active fragment is linked to cell survival and not to apoptosis. This result suggests that this phenomenon of active caspase-3 sequestration into the membrane compartment could inhibit apoptosis. Indeed, this phenomenon could be involved in the late apoptosis observed in neutrophils isolated from the inflammatory site of patients suffering from chronic inflammatory diseases, like rheumatoid arthritis.

To conclude, we have demonstrated that PR3 because of its particular subcellular localisation, not shared with its homologues, possesses protein target selectivity (procaspase-3 and p21 (4)) and biological functions, which could be involved in the regulation of neutrophil activity and survival . Therefore, PR3 should not be considered anymore as a simple proteinase released during neutrophil activation, which degrades extracellular matrix protein. We propose PR3 as a protein, which is able to regulate inflammatory process, in which its biological functions depend on its subcellular localisation . My work has highlighted mysterious PR3 features, which were hitherto unknown. PR3 as a simple serine protease homologue, strictly compartmentalised in granules, as common proteinases, surprises us by its biochemical aspects, its trafficking and finally by various targets, which can lead to different potential biological activities. Overall, the molecular and functional PR3 studies are linked to the molecular effector mechanisms of neutrophils, the effector cell of the inflammatory reaction . Therefore, the dogma that a cell in the last differentiation step is without any regulation in immunity, must be reconsidered.

 

In conclusion, a better understanding of the pro-inflammatory mechanisms and the process regulating neutrophil apoptosis, is essential to develop new therapeutic strategies by reducing neutrophils level, in chronic inflammatory diseases .

Magali Pederzoli, during her PhD, was supported by the “Association pour la Recherche sur la Polyarthrite Rhumatoïde” (ARP) and completed a forth year with a grant from the “Fondation pour la Recherche Médicale” (FRM).

Bibliography

1 -Durant S, Pederzoli M, Lepelletier Y, Canteloup S, Nusbaum P, Lesavre P, Witko-Sarsat V. Apoptosis-induced proteinase 3 membrane expression is independent from degranulation. J Leukoc Biol. 2004;75: 87-98.

2 - Kantari C*, Pederzoli-Ribeil M*, Amir-Moazami O, Cruz Moura I, Lecomte MC, Benhamou M, and Witko-Sarsat V. Proteinase 3, the Wegener autoantigen, is externalized during neutrophil apoptosis: evidence for a functional association with phospholipid scramblase 1 and interference with macrophage phagocytosis. * these two authors equally contributed to the work. Review in Blood

3 - Pederzoli M, Kantari C, Gausson V, Moriceau S, Witko-Sarsat V. Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: potential role of this compartmentalized activation of membrane-associated procaspase- 3 in neutrophils. J Immunol. 2005;174: 6381-90.

4 -Dublet B, Ruello A, Pederzoli M, Hajjar E, Courbebaisse M, Canteloup S, Reuter N, Witko-Sarsat V. Cleavage of p21/WAF1/CIP1 by proteinase 3 modulates differentiation of a monocytic cell line. Molecular analysis of the cleavage site. J Biol Chem. 2005; 280: 30242-53.

 

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