WO2007096388A1 - Polypeptides, pharmaceutical compositions and methods for the prevention and the therapeutic treatment of inflammatory disorders - Google Patents

Abstract

This invention is based on the experimental finding that a polypeptide derived from p47phox can be used as a therapeutical agent for treating or preventing the priming effect of GM-CSF and TNFα on neutrophil ROS production encountered in several inflammatory disorders. This invention concerns a polypeptide of 50 amino acids in length or less, comprising the following amino acid sequence: XPGPQZPGSPL (I) wherein X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and Z means an amino acid residue S or an amino acid residue T.

Description

Polypeptides, pharmaceutical compositions and methods for the prevention and the therapeutic treatment of inflammatory disorders.

Field of the invention The present invention relates to the field of the prophylaxis and the therapeutic treatment of inflammatory disorders, such as rheumatoid arthritis.

Background of the invention Inflammation occurs when tissues are injured during infection (by viruses, bacteria, fungi), trauma, chemicals, heat, cold or any other harmful stimulus. Chemicals including bradykinin, histamine, serotonin and others which are released, attracting from blood to tissues white blood cells especially neutrophils and monocytes to localize in an area to engulf and destroy foreign substances. During this process, chemical mediators called cytokines such as TNFα are released, giving rise to inflammation. Inflammatory disorders are those in which the inflammation is sustained or chronic. One example of an inflammatory disorder is rheumatoid arthritis. lmmunoinflammatory disorders (e.g., rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn's disease, stroke-induced brain cell death, ankylosing spondylitis, fibromyalgia, and autoimmune diseases such as asthma, multiple sclerosis, type I diabetes, systemic lupus erythematosus, scleroderma, systemic sclerosis, and Sjogren's syndrome) are inflammatory disorders characterized by dysregulation of the immune system and inappropriate mobilization of body's defenses against its own healthy tissue.

One percent of humans world-wide are afflicted with rheumatoid arthritis, a relentless, progressive disease causing severe swelling, pain, and eventual deformity and destruction of joints. According to the Arthritis Foundation, rheumatoid arthritis currently affects over two million Americans, of which women are three times more likely to be afflicted. Rheumatoid arthritis is characterized by inflammation of the lining of the joints and/or other internal organs, and the presence of elevated numbers of lymphocytes and high levels of proinflammatory cytokines. Treatment of immunoinflammatory disorders and, specifically, rheumatoid arthritis (RA) generally includes administration of (i) nonsteroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium salicylate); (ii) steroids (e.g., cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); (iii) DMARDs, i.e., disease modifying antirheumatic drugs (e.g., cyclosporine, azathioprine, methotrexate, leflunomide, cyclophosphamide, hydroxychloroquine, sulfasalazine, D-penicillamine, minocycline, and gold); or (iv) recombinant proteins (e.g., ENBREL® (etanercept, a soluble TNF receptor) and REMICADE® (infliximab) a chimeric monoclonal anti-TNF antibody). Thus, there is a recognized and permanent need in the art for new compounds which can be administered in order to prevent or treat inflammatory disorders.

Massive neutrophil accumulation at the inflammatory site and massive ROS release are believed to contribute to tissue injury in inflammatory disorders. ROS release can be potentiated by the presence of pro-inflammatory cytokines such as GM-CSF and TNFα at the inflammatory site.

For example, both excessive production of ROS and release of degradative enzymes by neutrophils have been implicated in rheumatoid tissue damage (4, 45). Consequently there also exists a need in the art for compounds that will prevent individuals from the consequences of the excess production of ROS due to an inflammatory disorder and, more generally, for treating patients having an inflammatory disorder. Particularly, in the definition of novel therapeutic compounds, avoiding exaggerated neutrophil responses in inflammatory environments while preserving the bacterial N-formyl peptides beneficial functions is searched.

Summary of the invention The present invention is based on the experimental findings that a polypeptide derived from the P47phox protein inhibits excessive production of reactive oxygen species (ROS) by neutrophils, due to an inflammatory disorders.

Accordingly, a first object of the invention consists of a polypeptide of 50 amino acids in length or less, comprising the following amino acid sequence:

XPGPQZPGSPL (I)

Wherein X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and Z means an amino acid residue S or an amino acid residue T.

In another aspect, the invention relates to a composition comprising :

(i) a polypeptide as defined above as an active ingredient

(ii) a mean for delivering said polypeptide in a cell. The invention also concerns pharmaceutical compositions comprising the polypeptides or compositions defined above.

In another aspect, the invention relates to an antibody directed against a polypeptide of formula :

QARPGPQS(phospho)PGSPLEEER (V), Wherein "S(phospho)" means a phosphorylated S amino acid residue. The invention concerns further, a method for the prevention or the treatment of an inflammatory disorder, comprising a step of administering a polypeptide, a composition or a pharmaceutical composition as defined above. The invention also concerns a method for detecting an inflammatory disorder in an individual, comprising the following steps :

(a) incubating a biological sample from said individual with an antibody as defined above, and

(b) measuring the amount of antibody which is bound to the P47phox protein, whereby said measured amount of said bound antibody is indicative of the presence or absence of an inflammatory disorder in said individual.

Description of the drawings Figure 1. Mass spectrometry analysis of GM-CSF-induced p47phox phosphorylation: serine 345 is phosphorylated in primed human neutrophils. (A) Data-dependent analysis of a tryptic peptide mixture, obtained using 4% of the SDS-PAGE band digested in situ, was performed during a 35-minutes gradient of 0-38% acetonitrile. The two upper panels depict the base peak chromatograms of MS/MS experiments performed during the analysis in information-dependent acquisition mode, and the lower panel represents the base peak chromatogram of the survey scans only. The arrow indicates the MS/MS scan of fragmented triply charged phosphopeptide. (B) Identification of the phosphorylated peptide QARPGPQ[pS]PGSPLEEER (amino acids 338-354). Tandem MS using collision-induced dissociation (CID) displayed a near-complete y-ion fragment ion series. Loss of phosphoric acid from the phosphoserine residue during CID, generating a dehydroalanine residue (69 Da), identified phospho-S345. Figure 2. Use of an antibody to phospho-serine 345 demonstrates that GM-CSF and TNFα induce phosphorylation of p47phox on Ser345 in a concentration- and time-dependent manner. (A) Neutrophils (1 x 10^ cells/ml) were incubated with various concentrations of GM-CSF for 20 minutes, or were incubated with GM-CSF (12.5 ng/ml) for the times indicated. Cells were lysed and proteins from 0.4 x 10⁶ cells were analyzed with SDS-PAGE and immunoblotting with anti-phospho-ser345 antibody (pSer345) or anti-p47phox antibody (p47phox). (B) Neutrophils

(1 x 10^ cells/ml) were incubated with various concentrations of TNFα for 20 minutes, or were incubated with TNFα (10 ng/ml) for the times indicated. Cells were lysed and proteins from 0.4 x 10⁶ cells were analyzed with SDS-PAGE and immunoblotting with anti-phospho-ser345 antibody (pSer345) or anti-p47phox antibody (p47phox). Data are representative of 3 independent experiments using cells from different donors.

Figure 3. Effect of different neutrophil agonists on the phosphorylation of Ser345. Neutrophils (1 x 10⁷ cells/ml) were incubated with GM-CSF (12.5 ng/ml) or TNFα (10 ng/ml) for 20 minutes, fMLP (10^"6 M) for 3 minutes, or PMA (100 ng/ml) for 10 minutes. Cells were then lysed and proteins from 0.4 x 10⁶ cells were analyzed with SDS-PAGE and immunoblotting with anti-phospho-ser345 antibody (pSer345) or anti-p47phox antibody (p47phox). Data are representative of 3 independent experiments using cells from different donors.

Figure 4 Effect of genistein, a protein tyrosine kinase inhibitor, on GM- CSF- and TNF-induced p47phox phosphorylation. Neutrophils were incubated without (control) or with 100 μM genistein (Genist.), for 30 minutes, then with 12.5 ng/ml GM-CSF (panel A) or 10 ng/ml TNFα for 20 minutes (panel B). P47phox from ³²P-labeled neutrophils (50 x 10⁶ cells) was immunoprecipitated with anti-p47phox antibody and analyzed by SDS-PAGE, Western blot and autoradiography ([³²P]p47phox). Total cell lysates (4 x 10⁵ cells) from unlabeled treated cells were also analyzed by SDS-PAGE and Western blot using the anti-phospho-Ser345 antibody (pSer345) or anti-p47phox antibody (p47phox). Data are representative of four experiments.

Figure 5 Effect of MAP-kinase inhibitors on GM-CSF- and TNF-induced p47phox and MAP-kinase activation. (A) Neutrophils (1 x 10⁷ cells/ml) were incubated with GM-CSF (12.5 ng/ml) or TNFα (10 ng/ml) for 20 minutes, then lysed, and proteins from 0.4 x 10⁶ cells were analyzed with SDS-PAGE and immunoblotting with anti-phospho-ERK1/2 or anti- phospho-p38 antibody. (B and C) Neutrophils were incubated with SB203580 (10 μM), PD98059 (50 μM) or UO126 (10 μM) for 30 minutes, then treated with (B) GM-CSF (12 ng/ml) or (C) TNFα (10 ng/ml) for 20 minutes. P47phox from ³²P-labeled neutrophils (50 x 10⁶ cells) was immunoprecipitated with anti-p47phox antibody and analyzed by SDS- PAGE, Western blot and autoradiography ([³²P]p47phox). Total cell lysates (4 x 10⁵ cells) from unlabeled GM-CSF- or TNFα-treated cells were also analyzed by SDS-PAGE, Western-blot with anti-phospho- Ser345 antibody (pSer345) or anti-phospho-ERK1/2 antibody (P-ERK1/2) which reflects MEK1/2 activity. P38 MAPK activity was assessed by in vitro phosphorylation of hsp27 as described in the Methods section. Data are representative of three experiments.

Figure 6. Effect of a cell-permeable peptide containing the Ser345- sequence on the priming effect of GM-CSF on neutrophil ROS production. Neutrophils were incubated with a cell-permeable peptide corresponding to amino acids 338-354 (ARPGPQSPGSPL) of p47phox (TAT-peptide-Ser345) or with a scramble peptide (TAT-peptide-Scramble) linked at the N-terminus to a highly basic peptide (YGRKKRRQRRR) derived from HIV tat protein, for 30 minutes, then with GM-CSF or TNF for 20 minutes, before stimulation with fMLP (10^~7 M). ROS production was measured with a luminol-amplified chemiluminescence technique. (mean +/- SEM, n = 4; * p< 0.05)

Figure 7. Priming of NADPH oxidase activity in neutrophils isolated from synovial fluid of patients with rheumatoid arthritis (RA). (A) Resting: Neutrophils (5 x 10⁵ cells) isolated from blood or from synovial fluid of RA patients were incubated in HBSS in the presence of luminol (10 μM), and spontaneous (without stimulation) chemiluminescence was measured over time. (B) The same preparation was then stimulated with fMLP (10^~7 M) and chemiluminescence was measured over time. Data are representative of 10 independent experiments using cells from different patients. (n=10 , p< 0.05, between synovial and blood neutrophils).

Figure 8. Phosphorylation of p47phox on Ser345, and phosphorylation of ERK1/2 and p38MAPK are increased in neutrophils isolated from synovial fluid of patients with rheumatoid arthritis (RA). Resting neutrophils (5 x 10⁵ cells) isolated from blood or from synovial fluid of RA patients were lysed and proteins were analyzed with SDS-PAGE and immunoblotting with anti-phospho-Ser345 antibody (pSer345), anti-phospho-ERK1/2 or anti-phospho-p38 antibody. Data are representative of 10 independent experiments using cells from different patients.

Figure 9. Effect of TAT-Ser345 peptide on NADPH oxidase activity of neutrophils from patients with rheumatoid arthritis

Neutrophils were incubated with a cell-permeable peptide corresponding to amino acids 338-354 (ARPGPQSPGSPL) of p47phox (TAT-peptide-

Ser345) [left panels] or with a scramble peptide (TAT-peptide-Scramble) linked at the N-terminus to a highly basic peptide (YGRKKRRQRRR) derived from HIV tat protein [right panels], for 30 minutes, before stimulation with fMLP (10^~7 M) [Fig. 7B] or without stimulation with fMLP [Fig. 7A]. ROS production was measured with a luminol-amplified chemiluminescence technique, (mean +/- SEM, n = 4; ^* p< 0.05)

Figure 10. Phosphorylation of p47phox on Ser345 and activation of ERK1/2 and p38 MAPK in neutrophils from patients with rheumatoid arthritis

Neutrophils from control patients and from three patients affected with rheumatoid polyarthritis were incubated. P47phox from ³²P-labeled neutrophils (50 x 10⁶ cells) was immunoprecipitated with anti-p47phox antibody and analyzed by SDS-PAGE, Western blot and autoradiography ([³²P]p47phox). Total cell lysates (4 x 10⁵ cells) were also analyzed by SDS-PAGE, and immunoblotting with anti-phospho-Ser345 antibody (pSer345), anti-phospho-ERK1/2 or anti-phospho-p38 antibody. Data are representative of 10 independent experiments using cells from different patients.

Figure 11. Mutation of Ser345 from p47phox into Ala inhibits NADPH oxydase hyperactivation induced by TNFα and GM-CSF in HL60 cells.

Fig. 11A : Undiifferenciated HL-60 cells (1 x 10⁷ cells/ml) [left panel] or neutrophil-differenciated HL-60 cells [right panel] were transfected with a cDNA encoding Ser345-p47phox (WT) or with a cDNA encoding the mutated S345A-p47phox (having an Alanine residue in position 345 instead of a Serine residue). Cells were then lysed and proteins from 0.4 x 10⁶ cells were analyzed with SDS-PAGE and immunoblotting with anti- p47phox antibody (p47phox) or anti-p67phox antibody (p67phox). Data are representative of 3 independent experiments using cells from different donors.

Fig. 11 B : Neutrophil-differentiated HL-60 cells were incubated with were transfected with a cDNA encoding Ser345-p47phox (WT) [left panel] or with a cDNA encoding the mutated S345A-p47phox (having an Alanine residue in position 345 instead of a Serine residue) [right panel], for 30 minutes, then with GM-CSF or TNFα for 20 minutes, before stimulation with fMLP (10^~7 M). ROS production was measured with a luminol- amplified chemiluminescence technique, (mean +/- SEM, n = 4; ^* p< 0.05)

Detailed description of the invention

It has been found according to the invention that a peptide derived from a neutrophil protein, specifically inhibits the priming effect of GM- CSF and TNFα on neutrophils ROS production. Neutrophils play a key role in host defenses against invading microorganisms and have a major role in inflammation (1 , 2, 3, 4). In response to a variety of agents, they release large quantities of superoxide anion (02 ^') and other ROS in a phenomenon known as the respiratory burst (4). Neutrophil production of 02 ^' is dependent on activation of NADPH oxidase, a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to 02 ^' (5, 6, 7). In resting cells NADPH oxidase is inactive and its components are distributed between the cytosol and membranes. When cells are activated, the cytosolic components (p47phox, p67phox, p40phox and Rac2) migrate to the membranes, where they associate with the membrane-bound component (flavocytochrome b558) to assemble the catalytically active oxidase (8-11). Upon NADPH oxidase activation, p47phox, p67phox, p40phox and p22phox become phosphorylated (12- 16). P47phox phosphorylation on several serines plays a pivotal role in oxidase activation in intact cells (17-19). As oxidants produced by NADPH oxidase are highly toxic not only for infectious agents but also for neighboring host tissues, tight regulation of the enzyme complex is necessary to control their production. Phosphorylation/dephosphorylation of the oxidase subunits is one such regulatory mechanism. Various kinases have been shown to phosphorylate p47phox in vitro, but the regulatory pathways involved in the in vivo priming and activation steps are unclear.

Neutrophil superoxide production can be potentiated by prior exposure to "priming" agents such as the pro-inflammatory cytokines GM- CSF, TNFα and IL-8 (20, 21 ). These cytokines inherently induce a very weak oxidative response by PMN, but they strongly enhance neutrophil release of ROS on exposure to a secondary applied stimulus such as bacterial N-formyl peptides (22, 23).

The intimate mechanisms involved in the priming process are poorly understood. Some studies have suggested that priming is regulated at the receptor and heterotrimeric G-protein levels (24, 25) or through an increase in cytochrome b558 expression (26-28). We and others have reported that priming of the human neutrophil respiratory burst by GM-CSF, LPS and TNFα is associated with partial phosphorylation of the cytosolic NADPH oxidase component p47phox (26, 29-31 ).

The GM-CSF receptor is composed of two chains designated α and β (32,33). Engagement of GM-CSF with its receptor activates a number of signal transduction pathways in human neutrophils, including PTK, PI3K (34-36) and the MAPK family ERK1/2 (37-40). GM-CSF binding to its receptor induces and increases the binding of the protein tyrosine kinase JAK2 and the src-tyrosine kinase lyn to the intracellular domain of the receptor (34, 41 , 42). These tyrosine kinases transduce the GM-CSF signal by phosphorylating other proteins such as STAT, PKB and PI3K. TNFα exerts its cellular effects by binding to its two receptors p55- and p75- TNFα-receptors. Neutrophils express both receptor types, (43) which are linked to different signaling pathways such as p38-MAPK (27,39). The molecular mechanisms by which GM-CSF and TNFα prime the respiratory burst in human neutrophils are not fully known.

It has been shown according to the invention that GM-CSF and TNFα induce p47phox phosphorylation on Ser345. It has also been shown according to the invention that in the respiratory burst priming conditions induced by GM-CSF and TNFa₁ phosphorylation of this site is markedly higher than in stimulatory conditions induced by the chemoattractant peptide fMLP or the protein kinase C activator PMA. The inventors have also shown that ERK1 /2 is the kinase involved in GM-CSF-induced phosphorylation of Ser345 and that p38MAPK is the kinase involved in TNFα-induced phosphorylation of Ser345. In addition, these phosphorylation pathways are activated in neutrophils isolated from synovial fluid of rheumatoid arthritis (RA) patients. All together, these results show that a peptide containing the

Ser345 sequence, specifically inhibits the priming effect of GM-CSF and TNFα on superoxide anion production by neutrophils in response to fMLP.

Since this priming effect is encountered in several inflammatory disorders, this peptide derived from p47phox can be used as a therapeutical agent for treating or preventing the priming effect of GM- CSF and TNFα on neutrophil ROS production encountered in several inflammatory disorders.

Furthermore, since this polypeptide specifically acts on the priming effects of cytokines on the neutrophil ROS production, this peptide does not modify the normal reaction of the neutrophil to a pathogen agent such as a bacteria. Accordingly, said polypeptide avoid exaggerated neutrophil responses in inflammatory environments while preserving the bacterial N- formyl peptides beneficial functions. Polypeptides according to the invention

A first object of the invention is a polypeptide of 50 amino acids in length or less, comprising the following amino acid sequence:

XPGPQZPGSPL (I) Wherein X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and Z means an amino acid residue S or an amino acid residue T.

The invention also concerns a polypeptide consisting of the following amino acid sequence: XPGPQZPGSPL (I)

Wherein X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and Z means an amino acid residue S or an amino acid residue T.

The term "polypeptide" as used herein refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L- amino acids joined by peptide bonds. The term "polypeptide" as used herein contain at least two amino acid residues and is less than about 50 amino acids in length.

In a preferred embodiment, the polypeptide defined above, comprises or consists of the following amino acid sequence :

AXPGPQZPGSPL (II), in which X and Z have the same meaning than in formula (I)

Preferably, in the polypeptide of formula (I), X means an amino acid residue R and Z means an amino acid residue S. In a preferred embodiment, the polypeptide of formula (I) consists of a polypeptide which possesses at least 11 consecutive amino acids of SEQ ID N°1 and comprises the amino acid sequence starting at the amino acid residue 338 and ending at the amino acid residue 354 of the p47phox protein of sequence SEQ ID N°1. Accordingly, a polypeptide as defined above possesses at least

11 , 12, 13, 14, 15, 20, 22, 25, 30, 35, 40, 45 or 49 consecutive amino acid of SEQ ID N°1 and comprises the amino acid sequence starting at the amino acid residue 338 and ending at the amino acid residue 354 of sequence SEQ ID N°1.

The term "protein" as used herein refers to a compound that is composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation. Proteins, as opposed to polypeptides, generally consist of chains of 50 or more amino acids.

By "p47phox" it is intended herein the protein of amino acid sequence SEQ ID N°1 and encoded by the nucleotide sequence SEQ ID N°2.

In a preferred embodiment, the polypeptide defined above, comprises or consists of the following amino acid sequence : ARPGPQSPGSPL (II). Several methods can be used for the synthesis of a polypeptide as defined above, including chemical synthesis, enzymatic method, or methods of genetic engineering.

With regard to the method of genetic engineering, polypeptide synthesis by the direct expression of polypeptides is extremely difficult since polypeptides are immediately subjected to digestion by proteases as they are expressed in the cells of microorganisms. For this reason, the fusion protein technique is currently the most commonly used technique in the gene recombination-based synthesis of polypeptides. In this technique, a polypeptide is expressed as a fusion protein fused to a protective protein, such as protein A, glutathione-S-transferase (GST), a polyhistidine tag, maltose binding protein (MBP) and beta-galactosidase, that can be readily separated by affinity chromatography and can thus allows rapid and efficient purification of the polypeptides. The desired polypeptide must then be excised from the fusion protein. Among known techniques used for this purpose are chemical methods, such as one involving the use of cyanogen bromide (which cleaves at the C-terminal side of Met), and enzymatic methods, by which proteins are cleaved immediately after the C-terminal ends of particular amino acid sequences such as enterokinase cleavage site (Asp-Asp-Asp-Asp-Lys) , thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser), preScission™ Protease (Amersham) cleavage site (Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro) and factor Xa cleavage site (Ile-Glu-Gly-Arg).

The tandem repeat technique can also be used. In this technique, genes of polypeptide of interest are linked in tandem within one molecule to be expressed as a stable precursor protein in the cells of microorganisms, and the polypeptides of interest are subsequently excised from the protein.

The polypeptide of formula (I) above, can also be produced by other methods known in the art. One method of producing the disclosed polypeptides fragment is to link two or more amino acid residues, or polypeptides together by protein chemistry techniques. For example, polypeptides are chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycar- bonyl) or Boc (tert-butyloxycarbonoyl) chemistry. A polypeptide can be synthesized and not cleaved from its synthesis resin, whereas the other fragment of a polypeptide can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group, which is functionally blocked on the other fragment. By polypeptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively. Alternatively, the polypeptide is independently synthesized in vivo.

Once isolated, these independent polypeptides may be linked to form a polypeptide of interest via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic polypeptide segments allow relatively short peptide fragments to be joined to produce larger polypeptide fragments, polypeptides or whole protein domains. The polypeptide of formula (I) defined above can be fused to another polypeptide able to insure the translocation of the polypeptide of formula (I) into a cell, specifically into a neutrophil.

As illustrated hereunder, a polypeptide able to insure the translocation of the polypeptide of formula (I) into a cell, specifically into a neutrophil can be a peptide derivated from the TAT protein from HIV, or from a peptide derived from the Antennapedia homeodomain from

Drosophila melanogaster.

Accordingly, in another embodiment, the polypeptide defined above is of formula :

RKKRRQRRR(α)_nXPGPQZPGSPL (III) wherein :

- n is an integer meaning 0 or 1 ,

- (α) means an amino acid sequence comprising 1 to 10 amino acids. - X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and

- Z means an amino acid residue S or an amino acid residue T.

The polypeptide of formula (III) comprises, from the N-terminal end to the C-Terminal end : a polypeptide derived from Tat and a polypeptide of formula (I) possibly linked together with a spacer (α). alternatively, in another embodiment, the polypeptide defined above is of formula :

YGRKKRRQRRR(α)_nXPGPQZPGSPL (I I Ia) wherein : - n is an integer meaning 0 or 1 ,

- (α) means an amino acid sequence comprising 1 to 10 amino acids.

- X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and

- Z means an amino acid residue S or an amino acid residue T. The polypeptides of formula (III) and (Ilia) comprise, from the N- terminal end to the C-Terminal end : a polypeptide derived from Tat and a polypeptide of formula (I) possibly linked together with a spacer (α).

Alternatively the polypeptide defined above is of formula : RQIKIWFQNRRMKWKK (α)_nXPGPQZPGSPL (IV) wherein :

- n is an integer meaning 0 or 1 ,

- (α) means an amino acid sequence comprising 1 to 10 amino acids.

- X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and

- Z means an amino acid residue S or an amino acid residue T.

The polypeptide of formula (IV) above comprises, from the N- terminal end to the C-Terminal end : a polypeptide derived from the Antennapedia homeodomain from Drosophila melanogaster and a polypeptide of formula (I), possibly linked together with a spacer α.

The attachment of the polypeptide of formula (I) defined above to a polypeptide able to insure the translocation of the polypeptide of formula (I) into a cell, in order to produce a fusion polypeptide of formula (III) or (IV), may be effected by any means which produces a link between the two constituents, which is sufficiently stable to withstand the conditions used and which does not alter the function of either constituent.

Preferably, the link between them, noted (α) above, is covalent. Numerous chemical cross-linking methods are known and potentially applicable for producing the fusion polypeptide of formula (III) or (IV). For example, non-specific chemical cross-linking methods, or preferably methods of direct chemical coupling to a functional group, found only once or a few times in one or both of the polypeptides to be cross-linked. Coupling of the two constituents can also be accomplished via a coupling or conjugating agent. There are several intermolecular cross- linking reagents, which can be used (see, for example, Means, G. E. et al. (1974)). Among these reagents are, for example, N-succinimidyl 3-(2- pyridyldithio) propionate (SPDP) or N, N'-(1 ,3-phenylene) bismaleimide.

Cross-linking reagents may be homobifunctional, i.e., having two functional groups that undergo the same reaction such as bismaleimidohexane ("BMH").

Alternatively, to solve the problems of polypeptide denaturation and contamination during chemical conjugation, recombinant techniques can be used to covalently attach the polypeptides of interest together, such as by joining the nucleic acid coding for the first polypeptide of interest with the nucleic acid sequence coding for the first polypeptide of interest and introducing the resulting gene construct into a cell capable of expressing the conjugate.

Recombinant methodologies required to produce a DNA encoding a desired polypeptide are well known and routinely practiced in the art. Laboratory manuals, for example MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor Press: Cold Spring Harbor, N. Y. (1989) describes in detail techniques necessary to carry out the required DNA manipulations. The fusion polypeptide can be produced in recombinant microorganism transformed therewith. In this process, each polypeptide component is preferably linked in the molecular ratio of 1 :1 (polypeptide of formula (I): polypeptide able to insure the translocation of the polypeptide of formula (I). The aid of an appropriate linker, designated (α) in the formula (III) and (IV) above, in order to allow proper folding of each polypeptide molecule can be useful. As a linker, it is preferable to use a peptide consisting of the appropriate number of amino acids to maintain activity of each polypeptide component, such as, a peptide composed of 0 to 20 amino acids, and preferably glycine, (glycine^ serine, or [(glycine^ serine]₂. Preferable vectors include any of the well known prokaryotic expression vectors, recombinant baculoviruses, COS cell specific vectors, or yeast-specific expression constructs.

Alternatively, the two separate nucleotide sequences of the polypeptides can be expressed in a cell or can be synthesized chemically and subsequently joined, using known techniques. Alternatively, the fusion polypeptide of formula (III) or (IV) can be synthesized chemically as a single amino acid sequence (i.e., one in which both constituents are present) and, thus, joining is not needed. To insure translocation or delivery into a cell of the polypeptides above, a vector can be used. Accordingly, the invention also relates to a composition comprising :

(i) a polypeptide as defined above as an active ingredient,

(ii) a mean for delivering said polypeptide in a cell. Preferably, the mean for delivering the polypeptide of formula (I) defined above, is selected from the group consisting of liposomes, a polymer of lysine, polymer of arginine, some toxins, or penetrating antibodies.

For example the one skilled in the art should use a detoxified exotoxin A (ETA) for delivering the polypeptide of formula (I) into a cell such as described in US patent application US6,086,900.

Alternatively, the one skilled in the art should use antibodies or their F(ab')2 and Fab' fragments as disclosed in International patent application WO 97/02840 which can penetrate into the interior of living cells, as immunovectors for intracytoplasmic transfer of the polypeptide of formula (I).

Pharmaceutical compounds according to the invention

It has been found according to the invention that a peptide derived from p47phox ^can be used as a therapeutic agent for treating or preventing the priming effect of GM-CSF and TNFα on neutrophil ROS production encountered in several inflammatory disorders.

Accordingly, the invention also concerns a pharmaceutical composition comprising a polypeptide or a composition as defined above and a pharmaceutically acceptable carrier.

Furthermore, since the peptide of formula (I) specifically acts on the priming effects of cytokines on the neutrophil ROS production, this peptide does not modify the normal reaction of the neutrophil to a pathogen agent such as a bacteria. Accordingly, a pharmaceutical composition comprising a polypeptide of formula (I) avoid exaggerated neutrophil responses in inflammatory environments while preserving the bacterial N-formyl peptides beneficial functions.

By "pharmaceutically acceptable carrier" is meant solid or liquid filler, diluent or substance which may be safely used in systemic or topical administration. Depending on the particular route of administration, a variety of pharmaceutically acceptable carriers well known in the art include solid or liquid fillers, diluents, hydrotropes, surface active agents, and encapsulating substances. As a general rule, the pharmaceutical composition of the present invention is conveniently administered orally, parenterally (subcutaneously, intramuscularly, intravenously, intradermal^ or intraperitoneally), intrabuccally, intranasally, or transdermally.

Pharmaceutically acceptable carriers for systemic administration that may be incorporated in the composition of the invention include sugar, starches, cellulose, vegetable oils, buffers, polyols and alginic acid. Specific pharmaceutically acceptable carriers are described in the following documents, all incorporated herein by reference: U.S. Pat. No. 4,401 ,663, Buckwalter et al. issued August 30, 1983; European Patent Application No. 089710, LaHann et al. published Sept. 28, 1983; and European Patent Application No. 0068592, Buckwalter et al. published Jan. 5, 1983. Preferred carriers for parenteral administration include propylene glycol, pyrrolidone, ethyl oleate, aqueous ethanol, and combinations thereof.

Representative carriers include acacia, agar, alginates, hydroxyalkylcellulose,hydroxypropyl methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, carrageenan, powdered cellulose, guar gum, cholesterol, gelatin, gum agar, gum arabic, gum karaya, gum ghatti, locust bean gum, octoxynol 9, oleyl alcohol, pectin, poly(acrylic acid) and its homologs, polyethylene glycol, polyvinyl alcohol, polyacrylamide, sodium lauryl sulfate, poly(ethylene oxide), polyvinylpyrrolidone, glycol monostearate, propylene glycol monostearate, xanthan gum, tragacanth, sorbitan esters, stearyl alcohol, starch and its modifications. Suitable ranges vary from about 0.5% to about 1 %. In a preferred embodiment, the pharmaceutical composition according to the invention, further comprises one or more components selected from the group consisting of surfactants, stabilizers, absorption promoters, water absorbing polymers, substances which inhibit enzymatic degradation, alcohol, organic solvents, oils, pH controlling agents, preservatives, osmotic pressure controlling agents, propellants, water and mixture thereof.

Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-Amine or NZ-Amine AS. Examples of emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil and other standard, metabolizable, non-toxic oils useful for injectables pharmaceutical compositions.

Conventional preservatives can be added to the pharmaceutical composition in effective amounts ranging from about 0.0001 % to about 0.1 % by weight. Depending on the preservative employed in the formulation, amounts below or above this range may be useful. Typical preservatives include, for example, potassium sorbate, sodium metabisulfite, phenol, methyl paraben, propyl paraben, thimerosal, etc.

For formulating a pharmaceutical composition according to the invention, the one skilled in the art will advantageously refer to the last edition of the European pharmacopoeia or of the United States pharmacopoeia.

Preferably, the one skilled in the art will refer to the fourth edition "2002" of the European Pharmacopoeia, or also to the edition USP 25- NF20 of the United States Pharmacopoeia. The weight amount of therapeutically active compound that is contained in each dose of the pharmaceutical composition of the invention will depend on the molecular weight of said therapeutically active compound as well as on the weight amount that is effective in preventing or inhibiting cellular phosphorylation of the amino acid residue serine in position 345 (serine 345) from the N-terminal end of the p47phox protein of amino acid sequence SEQ ID N°1.

For determining the appropriate amount of the therapeutically active compound, in a dose of a pharmaceutical composition of the invention, the one skilled in the art firstly determines the in vitro p47phox phosphorylation inhibiting ability of various weight amounts or concentrations of said therapeutically active compound, for example by using an antibody able to detect the phosphorylation state of serine 345 from p47phox protein. Such an antibody is described below. Then, the one skilled in the art will retain or select the given amount or concentration of said therapeutically active compound that blocks serine 345 phosphorylation from p47phox protein.

Then, the one skilled in the art transposes said retained or selected amount or concentration to the in vivo human situation, so that the concentration of said therapeutically active compound in the blood of a patient to which the pharmaceutical composition of the invention has been administered is identical to the concentration that blocks phosphorylation in vitro.

Methods of treatment according to the invention

The polypeptides, compositions, and pharmaceutical compositions defined above can be used for preventing or treating an inflammatory disorder.

Accordingly, the invention also concerns a method for the prevention or the treatment of an inflammatory disorder, comprising a step of administering a polypeptide, a composition or a pharmaceutical composition as defined above.

In a general manner, said inflammatory disorder is a disease where TNF inhibitors will be prescribed. Preferably, said inflammatory disorder is selected from the group consisting of inflammatory skin diseases such as psoriasis and dermatitis; responses associated with inflammatory bowel disease such as Crohn's disease and ulcerative colitis; ischemic reperfusion; adult respiratory distress syndrome; meningitis; encaphalitis; uveitis; autoimmune diseases such as rheumatoid arthritis, Sjogren's syndrome, vasculitis; diseases involving leukocyte diapedesis; multiple organ injury syndrome secondary to septicaemia or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex mediated diseases; hypovolemic shock; glomenulanephritis; multiple sclerosis; Type I diabetes melitis; acute and delayed hypersensitivity, graft vs. host disease; transplant rejection; reperfusion injury; endotoxic shock; disease states due to leukocyte dyscrasia and metastasis; asthma; pulmonary oxygen toxicity; inflammation of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, and cystic librosis, ulcerative colitis, stroke-induced brain cell death, ankylosing spondylitis, fibromyalgia, and autoimmune diseases such as asthma, multiple sclerosis, type I diabetes, systemic lupus erythematosus, scleroderma, and systemic sclerosis.

Most preferably, said inflammatory disorder is selected from the group consisting of rheumatoid arthritis, Crohn's disease and ankylosing spondylitis.

Antibodies according to the invention

The invention also deals with an antibody which can be used as a diagnostic tool to reveal the priming state of neutrophils in a patient and more generally, to reveal the inflammatory state of a patient.

It has been shown according to the invention that neutrophils isolated from patients having an inflammatory disorder are already primed to produce ROS, and this priming state is associated with upregulation of p47phox-Serine345 phosphorylation, ERK1/2 and p38MAPK phosphorylation in this inflammatory disorder.

In order to determine the priming state of neutrophils in inflammatory disorders, the inventors have isolated an antibody specifically directed against phosphorylated serine 345 of p47phox. Accordingly, the invention also relates to an antibody directed against a polypeptide of formula : QARPGPQS(phospho)PGSPLEEER (V),

Wherein "S(phospho)" means a phosphorylated Ser amino acid residue. It has been shown in the examples that this antibody is highly specific for phosphorylated p47phox, as it did not recognize other phosphorylated proteins.

Said anti-phosphorylated p47phox antibody may consist of a polyclonal antibody which may be obtained by (i) administering an immunologically effective amount of the purified polypeptide of formula

(V) above to an animal, preferably in combination with an adjuvant of immunity, such as albumin as shown in the examples below, (ii) then collecting the whole blood of the immunised animal and (iii) purifying the anti-phosphorylated p47phox polyclonal antibodies, such as for example by using an immunoaffinity chromatographic substrate onto which has previously been immobilised the purified polypeptide of formula (V). These techniques for obtaining purified polyclonal antibodies are well known from the one skilled in the art.

Said anti-phosphorylated p47phox antibody may consist of a polyclonal antibody, in which case said anti-phosphorylated p47phox antibody may be prepared from hybridomas obtained after fusion of B cells of animals immunised against the purified polypeptide of formula (V) with myeloma cells, according to the well known technique described by Kohler and Milstein in 1975.

Said anti-phosphorylated p47phox antibody may also consist of an antibody which has been produced by the trioma technique or by the human B-cell hybridoma technique described by Kozbor et al. in 1983.

Said anti-phosphorylated p47phox antibody may also consist of single chain Fv antibody fragments (United States Patent n° US

4,946,778; Martineau et al., 1998), of antibody fragments obtained through phage display libraries (Ridder at al., 1995) or of humanised antibodies (Reinmann et al., 1997; Leger et al., 1997).

Most preferably, the anti-phosphorylated p47phox antibody consists of a monoclonal antibody which is obtained by the following steps : (i) preparing a batch of purified polypeptide of formula (V);

(ii) immunising mice, for example BALB/c mice, with an effective amount of the purified polypeptide of formula (V) provided at step (ii), for example through three successive injection of said purified protein, each spaced by a one month time period, (iii) preparing hybridoma cell lines by fusion of the purified B cells of the mice immunised at step (ii), for example using the ClonaCell-HY hybridoma cloning kit according to the manufacturer's instructions (StemCell Technologies Inc., Vancouver, BC, Canada); (iv) culturing clones of the hybridoma cell lines prepared at step (iii) and selecting the clone(s) which secrete a monoclonal antibody directed against the phosphorylated p47phox protein; and

(v) purifying the monoclonal antibodies produced by the hybridoma cell clones which have been selected at step (iv).

For preparing a batch of purified polypeptide of formula (V) at step (i) of the method above, the one skilled in the art may perform one of the methods describe above in the part "polypeptides according to the invention".

The antibody defined above can be used in several diagnosis methods that will be described hereunder.

Diagnosis methods according to the invention

Another object of the invention is a method for detecting an inflammatory disorder in an individual, comprising the following steps : (a) incubating a biological sample from said individual with an antibody as defined above, and (b) detecting the binding of said antibody with the P47phox protein of sequence SEQ ID N°1 , wherein the binding of said antibody is indicative of the presence of an inflammatory disorder in said individual.

This method is particularly advantageous because it can be implemented completely in vitro on a blood sample from an individual. The detection of an inflammatory disorder consists of raw experimental data indicative of the phosphorylation status of the p47phox protein from a tested patient, since, as already mentioned above, there is a relevant correlation between (a) the phosphorylation of p47phox on Ser 345 and (b) an inflammatory disorder in which neutrophils are primed to produce ROS. The sole detection of the phosphorylation state of p47phox on Ser345 might not be sufficient for a global accurate clinical diagnosis, or prognosis, of an inflammatory disorder within the patient tested. Thus the measurement of the expression level of the phosphorylation state of p47phox on Ser345 might be completed by, or combined with, other diagnosis or prognosis markers of an inflammatory disorder.

For performing the detection of anti-p47phox antibody bound to the P47phox protein, the one skilled in the art can follow the procedures described in the part "Material and Methods" below. For example, for performing said detection, neutrophils can be isolated from a blood sample of a patient, the neutrophils are then lysed and the cell lysate obtained is incubated with the anti-p47 phox antibody described above. The p47phox protein phosphorylated on ser345 bound to the antibody is then identified by SDS-polyacrylamide gel electrophoresis.

In a general manner, said inflammatory disorder is a disease where TNF inhibitors will be prescribed.

The inflammatory disorders that can be detected according to the method above are selected from the group consisting of psoriasis and dermatitis, Crohn's disease, ulcerative colitis, ischemic reperfusion; adult respiratory distress syndrome; meningitis; encaphalitis; uveitis; rheumatoid arthritis, Sjogren's syndrome, vasculitis; diseases involving leukocyte diapedesis; multiple organ injury syndrome secondary to septicaemia or trauma; alcoholic hepatitis; bacterial pneumonia; hypovolemic shock; glomenulanephritis; multiple sclerosis; Type I diabetes melitis; acute and delayed hypersensitivity, graft vs. host disease; transplant rejection; reperfusion injury; endotoxic shock; disease states due to leukocyte dyscrasia and metastasis; asthma; pulmonary oxygen toxicity; pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, and cystic librosis, stroke-induced brain cell death, ankylosing spondylitis, fibromyalgia, systemic lupus erythematosus, scleroderma, and systemic sclerosis.

EXAMPLES Material and Methods

Reagents and antibodies.

Recombinant human GM-CSF and TNFα were from R&D Systems and

Peprotech-Tebu France. FMLP, PMA, protease and phosphatase

32 inhibitors were from Sigma Chemical Co. P-orthophosphoric acid was from NEN-Dupont. Kinase inhibitors were from Calbiochem. Injection- grade water and 0.9% NaCI were endotoxin-free (<0.4 pg/ml) in the limulus test (Charles River, Charlestone, USA). Endotoxin-free buffers and salt solutions were from Invitrogen. Rabbit polyclonal antibody against p47phox was a generous gift from Dr B. M. Babior (The Scripps Research Institute, CA, USA). For the production of an antibody directed against phospho-serine 345, rabbits were injected with the ovalbumin- crosslinked phosphopeptide sequence of p47phox

(QARPGPQS(phospho)PGSPLEEER), produced by Neosystem (Strasbourg, France). The peptides used in this study, corresponding to amino acids 338-354 (ARPGPQSPGSPL) of p47phox (TAT-peptide- Ser345) and a control scramble peptide (PRGLAPPSGQPS) (TAT- peptide-Scramble) linked at the N-terminus to a highly basic peptide (YGRKKRRQRRR) derived from HIV tat protein, were synthesized by Neosystem (Strasbourg, France).

Neutrophil preparation and p47phox immunoprecipitation. Human neutrophils were obtained in LPS-free conditions by means of Dextran sedimentation and Ficoll centrifugation as previously described (29). Neutrophils in HBSS (25 x 10⁶ /ml) were treated with GM-CSF (12 ng/ml) or TNFα (10 ng/ml) at 37°C for 20 minutes which correspond to optimal conditions to prime othe respiratory burst in response to fMLP (29,30). The reaction was stopped by adding ice-cold buffer and by centrifugation at 400 g for 6 minutes at 4°C. The cells were lyzed by resuspension in lysis buffer (20 mM Tris-HCI pH 7.4, 150 mM NaCI, 0.25 M sucrose, 5 mM EGTA, 5 mM EDTA, 15 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1.5 mM PMSF, 1 mM DFP, 0.5% Triton X- 100, 25 mM NaF, 5 mM NaVO₄, 5 mM β-glycerophosphate, 1 mM pNPP, 1 mg/ml DNase I) and sonication on ice (3 x 15 s); the lysate was then centrifuged at 100 000 g for 30 minutes at 4°C in a TL100 ultracentrifuge (Beckman Inc.). The cleared supernatant was incubated overnight with anti-p47phox (1/200) and protein was immunoprecipitated with Gamma- bind G-sepharose beads (Pharmacia), washed four times in lysis buffer, and denatured in Laemmli's sample buffer. The samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 10% polyacrylamide gels, using standard techniques (58). The separated proteins were stained with Coomassie blue and the protein band corresponding to p47phox was cut out, treated with protease, and analyzed by mass spectrometry as described below.

Preparation of p47phox samples, and mass spectrometry. Automated Nano-flow LC-MS/MS Analysis:

P47phox was isolated by SDS PAGE, stained with Coomassie blue and digested in situ with trypsin and LysC. Extracted peptide mixtures were analyzed with a nanoflow capillary high-pressure liquid chromatography system (Ultimate, LC-Packings) directly coupled to a Q-TOF tandem mass spectrometer (Q-TOF Ultima API, Waters/Micromass, Manchester, UK) as described elsewhere (46). Peptide mixtures were separated on a 9-cm custom-packed C18 reverse-phase column (75 μm ID, Zorbax SB- C18, 3.5 μm particle size) during a 35-min gradient of 0-90% acetonitrile (v/v) containing 1 % (v/v) formic acid, 0.6% (v/v) acetic acid, and 0.005% (v/v) heptafluorobutyric acid at a flow rate of 175 nl/min. The mass spectrometer was calibrated with NaI.

Mass spectra obtained by automated nano-flow LC-MS/MS and data-dependent acquisition modes were analyzed with MassLynx 3.5 software (Waters Corporation, UK) and Mascot MS-MS Ions Search software (Matrixscience, UK). Tandem mass spectra obtained with phosphopeptides were manually inspected and compared to theoretical data by obtained using the GPMAW 6.20 program (Lighthouse Data, Odense, Denmark).

32p-iabeling of neutrophils; stimulation and fractionation.

Cells were incubated in phosphate-free buffer (20 mM Hepes pH 7.4, 140 mM NaCI, 5.7 mM KCI, 0.8 mM MgCI₂ and 0.025% BSA) containing 0.5 mCi 32p__orthophosphoric acid/1 O^ cells/ml for 60 minutes at 30⁰C, as previously reported (29). Cells were then treated with GM-CSF or TNFα, as described above. The reaction was stopped by adding ice-cold buffer and centrifugation at 400 g for 6 minutes at 4°C. The cells were lysed and p47phox was immunoprecipitated as described above. The samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 10% polyacrylamide gels, using standard techniques. The separated proteins were transferred to nitrocellulose and detected as described elsewhere

(29).

Preparation of neutrophils from synovial fluid and peripheral blood from RA patients.

With the subject's informed consent, knee-joint synovial fluid and peripheral blood were collected from 6 patients who met the American College of Rheumatology criteria for RA (59,60) and who were never received any anti-cytokine biotherapy. Samples were collected in sterile lithium heparinate tubes, placed on ice and transferred immediately to the laboratory. After centrifugation of synovial fluid at 400 g for 15 minutes at 4°C, neutrophils were isolated from the pellet by washing with PBS and Ficoll centrifugation. Blood neutrophils were isolated from RA patients and in parallel from healthy donors by Dextran sedimentation and Ficoll centrifugation as described above (29). In some instances, after the samples collection in sterile lithium heparinate tubes, and in order to make sure that neutrophil preparation was performed in the same conditions and at the same time, synovial fluid and venous blood was diluted twice in sterile PBS and cells were isolated by one centrifugation step using a polymorphprep gradient (Abcys SA, Axis-Shiekd). After centrifugation at 500 g for 30 minutes at 22°C, the neutrophil band was collected and cells washed in PBS and counted, then used for ROS production and Western blots.

Measurement of O2 ^~ and ROS production. Then 02 ^' production was measured in response to fMLP (10^~7 M) in terms of superoxide dismutase-inhibitable (SOD) ferricytochrome c reduction as described elsewhere (29), or chemiluminescence method was used for added sensitivity: cells (2.5 x 10⁵) were suspended in 0.5 ml HBSS containing 10 μM luminol preheated to 37⁰C in the thermostated chamber of the luminometer (Berthold-Biolumat LB937) and allowed to stabilize. After a baseline reading, cells were stimulated with 10^~7 M fMLP and chemiluminescence was recorded.

Detection of Ser345 phosphorylation in neutrophils by a specific antibody. Neutrophils (1 x 10⁷ cells/ml) were incubated with various concentrations of GM-CSF, TNF, FMLP or PMA or at different incubation times. Cells were lysed in the lysis buffer described above and proteins from 0.4 x 106 cells were analyzed with SDS-PAGE and immunoblotting with anti- phospho-ser345 antibody or anti-p47phox antibody and HRPO-labeled- goat anti-rabbit antibody. The reaction was detected using ECL reagents. HL-60 cells culture, transfection and differentiation. HL-60 cells were purchased from American Type Culture Collection (ATCC) cultured in RPMI 1640 containing 10% heat inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in humidified atmosphere, 5% CO2 in air. Plasmids (pEBO) that encodes wild type (WT) p47phox or p47phox in which serine 345 was mutated to alanine (S345A) were constructed (gift from Dr B. Babior). HL-60 cells were transfected by electroporation. After 48 hours hygromycin B was added to a final concentration of 200 μg/ml and the cells were grown under hygromycin B selection pressure for 2 weeks. P47phox-protein expression in transfected HL60 cells was monitored by Western blot technique. For differentiation, exponentially growing cells were harvested and resuspended at density of 5 x 10⁵ cells/ml in complete medium with hygromycin B in the presence of 1.3% DMSO. After 5 days, the cells were washed and incubated for 5 hours in RPMI alone, then assessed for NADPH oxidase activity using chemiluminescence.

Statistical analysis All results are expressed as means ± standard error of the mean

(SEM). Significant differences were identified with Student's t test; p values of < 0.05 were considered significant.

Example 1 : Identification of the GM-CSF- and TNFα-induced p47Phox phosphorylation target site.

In fMLP- or PMA-stimulated human neutrophils, p47phox is phosphorylated on several serines located between Ser303 and Ser379 (17, 18). In contrast, in GM-CSF- or TNFα-primed human neutrophils, p47phox is partially phosphorylated on a common peptide which could contain several serines as potential targets of these cascades (29, 30). In order to identify the p47phox site(s) which is (are) phosphorylated upon GM-CSF or TNFα exposure, we immunopurified p47phox from GM-CSF- and TNFα-treated cells (500 x 10⁶) and analyzed it by mass spectrometry. The protein was digested with trypsin and endoproteinase Lys-C, and the resulting peptide mixtures were analyzed by LC-ESI- MS/MS with data-dependent acquisition. The phosphopeptide QARPGPQ[pS]PGSPLEEER (amino acids 338-354) was detected at 26 min and was automatically selected for sequencing by MS/MS (Figure 1 ) (The same result was obtained with GM-CSF and TNFα, but only the GM- CSF result is shown here). The presence of a phosphoserine at position 345 was revealed by MS/MS, based on the mass difference of 69 Da (dehydroalanine) between the y9 and y10 fragment ions (Figure 1-B). Dehydroalanine is generated upon phosphoric acid elimination from phosphoserine, and is commonly observed in MS/MS (46).

Example 2: Use of an antibody to phosphoserine 345 demonstrates that GM-CSF and TNFαinduce Ser345 phosphorylation.

To examine Ser345 phosphorylation status in cellular extracts, we prepared a polyclonal antibody directed against phospho-serine 345 by using as antigen the peptide QARPGPQ[pS]PGSPLEEER (p47phox amino acids 338-354). Western blot analysis of whole lysates from GM- CSF- or TNFα-treated neutrophils (from as low as 4 x 10⁵ cells) showed (Figure 2-A and 2-B (pSer345)) that this antibody was highly specific for phosphorylated p47phox, as it did not recognize other phosphorylated proteins. GM-CSF- and TNFα-induced p47phox phosphorylation at serine 345 were both concentration-dependent and time-dependent, and closely matched the priming effects of GM-CSF and TNFα on superoxide production (29,30). Specific detection of p47phox with an antibody that recognizes the non phosphorylated as well as the phosphorylated protein (Figure 2-A and B-p47phox) showed that approximately the same amount of p47phox was present in each sample.

We next examined whether other agents known to activate neutrophils triggered Ser345 phosphorylation. We found that phosphorylation of this site was induced more potently by the proinflammatory cytokines GM- CSF and TNFα which induce the priming process, than by neutrophil- stimulating agents such as the chemoattractant peptide formyl-methionyl- leucyl-phenylalanine (fMLP) and the protein kinase C activator phorbol myristate acetate (PMA) used at 10^~6 M and 100 ng/ml, respectively (optimal concentration triggering the respiratory burst) (Figure 3). Specific detection of p47phox with an antibody that recognizes the non phosphorylated as well as the phosphorylated protein showed that the same amount of p47phox was present in each sample. These results suggest that serine 345 is more strongly phosphorylated during GM-CSF or TNFα-induced priming than in stimulatory conditions.

Example 3: Effect of tyrosine kinase and MAP-kinase inhibitors on GM-CSF- and TNFα-induced p47phox phosphorylation.

Myeloid cells such as neutrophils are terminally differentiated short-lived cells and are resistant to transfection. An alternative strategy to study the role of specific enzymes is to use cell-permeant pharmacologic inhibitors. In previous studies (29,30) we showed, by ³²P-labeling, that the broad- range protein tyrosine kinase inhibitor genistein inhibited GM-CSF- and TNFα-induced p47phox phosphorylation. To verify that the Ser345 phosphorylation detected with the specific antibody is controlled by the same upstream tyrosine kinases, we tested the effects of genistein on this process. First, p47phox from ³²P-labeled neutrophils (50 x 10⁶ cells) was immunoprecipitated with anti-p47phox and analyzed by SDS-PAGE, Western blotting and autoradiography (Figure 4A+B, (³²P)p47phox). Second, total cell lysates (4 x 10⁵ cells) were analyzed by SDS-PAGE and Western-blot with the anti-phospho-Ser345 antibody (Figure 4A+B, pSer345). Both methods showed that genistein inhibited GM-CSF- and TNFα-induced p47phox phosphorylation, confirming that the anti- phospho-Ser345 antibody recognizes the phosphorylated site on p47phox induced by GM-CSF and TNFα and that a genistein sensitive tyrosine kinase controls this event.

As serine 345 is located in a sequence recognized and phosphorylated by MAPKinase (-PXSP-), we examined which MAP-kinase was involved in GM-CSF- and TNFα-induced p47phox phosphorylation at Ser345 by testing the effects of different MAPK inhibitors. As GM-CSF and TNFα induce the activation of ERK1/2 and p38 MAPK, respectively, in suspended human neutrophils (Figure 5-A and ref 39, 40, 47), we used PD98059 and UO126, which inhibit MEK1/2 (the upstream activator of ERK1/2) (48, 49) and also SB203580, a p38 MAPK inhibitor (50), to analyze the role of these two MAPK pathways in p47phox phosphorylation in intact neutrophils. Neutrophils were incubated for 45 min with PD98059 (50 μM), UO126 (10 μM) or SB203580 (10 μM), then treated with GM-CSF or TNFα. P47phox phosphorylation status was analyzed by comparing the results of the two methods (³²P-labeling and anti-phosphoSer345 antibody). As shown in Figure 5B, PD98059 and UO126 abrogated p47phox phosphorylation on Ser345 in parallel with the inhibition of ERK1/2 phosphorylation (Figure 5, p-ERK) induced by GM- CSF. SB203580 had no inhibitory effect, it rather moderately enhanced GM-CSF-induced phosphorylation of p47phox and ERK1/2, an effect not further examined here. This strongly pointed to ERK1/2 as the kinases involved in p47phox phosphorylation on serine 345 in GM-CSF-primed human neutrophils. However, with regard to TNFα-treated neutrophils, the p38MAPK inhibitor SB203580 inhibited phosphorylation of p47phox on Ser345, but the MEK1/2 inhibitors (PD98059 and U0126) had no effects. In these conditions SB203580 inhibited p38MAPK activity, as shown by inhibition of the phosphorylation of hsp27, a known substrate of MAPK-activated kinase 2, which is a downstream target of p38MAPK. In all these experiments, specific detection of p47phox, ERK1/2 and hsp27 with specific antibodies that recognize the non phosphorylated as well as the phosphorylated proteins showed that the same amounts of each protein were present in each sample (Figure 5 A+B and data not shown). All these results suggest that ERK1/2 are the kinases involved in p47phox phosphorylation on serine 345 in GM-CSF-primed human neutrophils, while p38MAPK is the kinase involved in p47phox phosphorylation on serine 345 in TNFα-primed human neutrophils. These two pathways could be redundant for the phosphorylation of p47phox on this specific site.

Example 4: A cell-permeable peptide containing the Ser345- sequence specifically inhibits the priming effect of GM-CSF and TNFα on neutrophil ROS production.

As stated above, neutrophils are terminally differentiated short-lived cells that are resistant to transfection. An alternative strategy to study the role of specific proteins or enzymes in their functions is to use cell-permeant pharmacologic inhibitors or cell-permeant peptides. To determine whether phosphorylation of Ser345 is directly linked to the priming process in intact cells, we generated an inhibitory cell-permeable peptide (TAT- peptide-Ser345) corresponding to amino acids 338-354 (ARPGPQSPGSPL) of p47phox, linked at the N-terminus to a highly basic peptide (YGRKKRRQRRR) derived from HIV tat protein. This permits peptide translocation across cell membranes (51 ), and provides a very effective protein delivery method in human neutrophils (52). This peptide is expected to compete with the Ser345 of p47phox, as a substrate for ERK1/2 and p38MAPK. Cells were first treated with the peptide for 30 min and then with GM-CSF or TNFα for 15 min before fMLP stimulation. For greater sensitivity, ROS production was measured in terms of luminol-amplified chemiluminescence. As shown in Figure 6, tat-p47phox(338-354) inhibited both GM-CSF- and TNFα-induced priming of the respiratory burst, in a concentration-dependent manner, while the tat-scramble peptide had little or no inhibitory effect. These results suggest that Ser345 plays a critical role in the priming of neutrophil superoxide production induced by GM-CSF and TNFα.

Example 5: Priming of NADPH oxidase activity and phosphorylation of p47phox on Ser345 in neutrophils from patients with rheumatoid arthritis (RA).

Massive neutrophil accumulation at the inflammatory site and massive ROS release are believed to contribute to tissue injury in inflammatory diseases. ROS release can be potentiated by the presence of proinflammatory cytokines such as GM-CSF and TNFα at the inflammatory site. Because pro-inflammatory cytokines are involved in several inflammatory diseases such as RA, and anti-TNFα therapies are very beneficial in this disease, we examined NADPH oxidase priming, p47phox-Ser345 phosphorylation and ERK1/2 or p38MAPK phosphorylation status in neutrophils from patients with RA. The results showed (Figure 7-A) that neutrophils from synovial fluid of RA patients had higher basal NADPH oxidase activity than neutrophils isolated from blood of the same patient. When stimulated with fMLP, neutrophils from synovial fluid of RA patients had higher ROS production than circulating neutrophils (Figure 7-B) suggesting that they are primed. Furthermore, phosphorylation of p47phox on Ser345, phosphorylation of ERK1/2 and phosphorylation of p38MAPK were all increased in RA patients' synovial neutrophils as compared to their blood neutrophils (Figure 8). Taken together, these results suggest that resident synovial neutrophils from RA patients are in a primed state and that phosphorylation of p47phox on Ser345 by redundant kinases such as ERK1/2 and p38MAPK could be the molecular basis of this process. Example 6: The cell-permeable peptide TAT-Ser345 inhibits the basal and primed ROS production by neutrophils from RA patients.

The effect of the TAT-Ser345 inhibitor peptide on the hyperactivation of the neutrophils NADPH oxidase from patients affected with rheumatoid arthritis was tested.

The results depicted in Figure 9 show that the TAT-345 peptide effectively inhibits the spontaneous neutrophil activity, as well as the hyperactivation which is induced in vivo in the RA patients.

Example 7: phosphorylation of p47phox on Ser345 and activation of

MAPKinases in neutrophils from patients with rheumatoid arthritis

(RA).

The anti-phospho-Ser345 antibody has been tested on further RA patients.

The results depicted in Figure 10 show that phosphorylation of p47phox on Ser345, phosphorylation of ERK1/2 and phosphorylation of p38MAPK were all increased in RA patients' synovial neutrophils as compared to synovial neutrophils from control patients.

Example 8: Mutation of Ser345 to Ala inhibits the priming process in

HL-60 cells.

In view of showing the role of Ser345 from p47phox in the priming of neutrophils, a mutated S345A p47phox has been generated. The cDNA encoding the mutated S345A p47phox was transfected in HL-60cells wich have then been differentiated towards neutrophils and then primed with

TNFα and GM-CSF.

The results depicted in Figure 11 show that the NADPH oxidase hyperactivation of HL-60 cells transfected with the mutated S345A p47phox was inhibited. Those results fully confirm the involvement of the phosphorylation of the Ser345 residue from p47phox in the priming of the NADPH hyperactivation of the neutrophils.

References

1. Babior, B. M. 2000. Phagocytes and oxidative stress. Am. J. Med. 109:33-44.

2. Klebanoff, S.J. 2005. Myeloperoxidase: friend and foe. J. Leuk. Biol. 77:598-625.

3. Segal, A.W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. 23: 197-223. 4. Babior, B. M. 1984. Oxidants from phagocytes: agents of defense and destruction. Blood. 64:959-966.

5. Chanock, S.J., El-Benna, J., Smith, R.M., and Babior, B. M. 1994. The respiratory burst oxidase. J. Biol. Chem. 269:24519-24522.

6. Quinn, M.T., Gauss, K.A. 2004. Structure and regulation of the neutrophil respira tory burst oxidase: comparison with nonphagocyte oxidases. J. Leukoc. Biol. 76:760-781.

7. Groemping, Y., Rittinger, K. 2005. Activation and assembly of the NADPH oxidase: a structural perspective. Biochem J. 386:401- 416. 8. Clark, R. A., Volpp, B. D., Leidal, K. G., and Nauseef, W.M. 1990.

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J. Clin. Invest. 85:714-721.

9. Quinn, M.T., Evans, T., Loetterle, L. R., Jesaitis, A.J., and Bokoch, G. M. 1993. Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. J. Biol. Chem. 268:20983-20987.

10.AbO, A., Webb, M. R., Grogan, A., and Segal, A.W. 1994.

Activation of NADPH oxidase involves the dissociation of p21 rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. Biochem. J.

298:585-591. 11. El Benna, J., Ruedi, J. M., and Babior, B. M.1994. Cytosolic guanine nucleotide-binding protein Rac 2 operates in vivo as a component of the neutrophil respiratory burst oxidase, transfer of

Rac 2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. J.Biol.Chem. 269:6729-6734.

12.0kamura, N., Curnutte, J.T., Roberts, R.L, and Babior B. M. 1988. Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 47-kDa proteins in two forms of chronic granulomatous disease. J. Biol. Chem.

263:6777-6782. 13. Dusi, S., and Rossi, F. 1993. Activation of NADPH oxidase of human neutrophils involves the phosphorylation and the translocation of cytosolic p67phox. Biochem. J. 296:367-371. 14. EI-Benna, J., Dang, P.M.C., Gaudry, M,. Fay, M., Morel, F.,

Hakim, J., and Gougerot-Pocidalo M.A. 1997. Phosphorylation of the respiratory burst oxidase subunit p67phox during human neutrophil activation. Regulation by protein kinase C-dependent and independent pathways. J. Biol. Chem. 272:17204-17208. 15. Bouin, A.P., Grandvaux, N., Vignais, P.V., Fuchs, A. 1998. p40(phox) is phosphorylated on threonine 154 and serine 315 during activation of the phagocyte NADPH oxidase. Implication of a protein kinase c-type kinase in the phosphorylation process. J.

Biol. Chem. 273:30097-30103. 16. Regier, D.S., Greene, D. G., Sergeant, S., Jesaitis, A.J., McPhail,

L.C. 2000. Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation. J. Biol. Chem. 275:28406-288412. 17. El Benna, J., Faust, L. P., and Babior, B. M. 1994. The phosphorylation of the respiratory burst oxidase component p47phox during neutrophil activation. Phosphorylation of sites recognized by protein kinase C and by proline-directed kinases. J.

Biol. Chem. 269:23431-23436. 18. El Benna, J., Faust, L. P., Johnson, J. L., and Babior, B. M. 1996.

Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping.

Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase. J. Biol. Chem. 271 :6374-6378.

19. Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S.J. 1995. The phosphorylation targets of p47phox, a subunit of the respiratory burst oxidase. Functions of the individual target serines as evaluated by site-directed mutagenesis. J. Clin. Invest. 96:1499-1505.

20. Downey, G. P., Fukushima, T., Fialkow, L. and Waddell, T.K. 1995. Intracellular signaling in neutrophil priming and activation. Semin. Cell. Biol. 6:345-356.

21. Hallett, M. B., and Lloyds, D. L. 1995. Neutrophil priming: the cellular signals that say 'amber' but not 'green', immunol. Immunol. Tod. 16:264-268.

22. Elbim, C, Bailly, S., Chollet-Martin, S., Hakim, J., and Gougerot- Pocidalo, M.A. 1994. Differential priming effects of proinflammatory cytokines on human neutrophil oxidative burst in response to bacterial N-formyl peptides. Infect. Immun. 62:2195-

2201.

23. Elbim, C, Chollet-Martin, S., Bailly, S., Hakim, J., and Gougerot- Pocidalo, M.A. 1993. Priming of polymorphonuclear neutrophils by tumor necrosis factor alpha in whole blood: identification of two polymorphonuclear neutrophil subpopulations in response to formyl-peptides. Blood. 82:633-640.

24. Mc Coll, S. R., Beauseigle, D., Gilbert, C, and Naccache, P. H. 1990. Priming of the human neutrophil respiratory burst by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha involves regulation at a post-cell surface receptor level. Enhancement of the effect of agents which directly activate G proteins. J. Immunol. 145:3047-3053.

25. KeN, M. L., Solomon, N. L., Lodhi, I.J., Stone, K.C., Jesaitis, A.J., Chang, P.S., Linderman, J.J., Omann, G. M. 2003. Priming- induced localization of G(ialpha2) in high density membrane microdomains. Biochem. Biophys. Res. Commun. 301 :862-872.

26. DeLeo, F. R., Renee, J., McCormick, S., Nakamura, M., Apicella, M., Weiss, J. P., and Nauseef, W.M. 1998. Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J. Clin. Invest. 101 :455-463.

27. Ward, R.A., Nakamura, M., McLeish, K.R. 2000. Priming of the neutrophil respiratory burst involves p38 mitogen-activated protein kinase-dependent exocytosis of flavocytochrome b558-containing granules. J. Biol. Chem. 275:36713-36719.

28. Mansfield, P.J., Hinkovska-Galcheva, V., Shayman, J.A., Boxer, L.A. 2002. Granulocyte colony-stimulating factor primes NADPH oxidase in neutrophils through translocation of cytochrome b(558) by gelatinase-granule release. J. Lab. CHn. Med. 140:9-16. 29. Dang, M. P-C, Dewas, C, Gaudry, M., Fay, M., Gougerot-

Pocidalo, and El Benna, J. 1999. Priming of human neutrophil respiratory burst by granulocyte/macrophage colony-stimulating factor (GM-CSF) involves partial phosphorylation of p47phox. J. Biol. Chem. 274:20704-20708. 30. Dewas, C, Dang, PMC, Gougerot-Pocidalo, M-A., and El Benna

J. 2003. TNF-alpha induces phosphorylation of p47(phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte-macrophage colony-stimulating factor. J. Immunol.

171 :4392-4398. 31. Brown, G. E., Stewart, M.Q., Bissonnette, S.A., ENa, A.E., Wilker,

E., Yaffe, M. B. 2004. Distinct ligand-dependent roles for p38

MAPK in priming and activation of the neutrophil NADPH oxidase.

J. Biol. Chem. 279:27059-27068.

32. Miyajima, A., Mui, A.L., Ogorochi, T., Sakamaki, K. 1993. Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood. 82:1960-1974. 33. Bagley, CJ. , Woodcock, J. M., Stomski, F.C., Lopez, A.F. 1997.

The structural and functional basis of cytokine receptor activation: lessons from the common beta subunit of the granulocyte- macrophage colony-stimulating factor, interleukin-3 (IL-3), and IL-

5 receptors. Blood. 89:1471-1482. 34. Corey, S., Eguinoa, A., Puyana-Theall, K., Bolen, J. B, Cantley, L.,

Mollinedo, F, Jackson, T.R, Hawkins PT. , and Stephens, L. R.

1993. Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide

3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells. EMBO J. 12:2681-2690. 35.AI-Shami, A., Naccache, P. H. 1999. Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Involvement of Jak2 in the stimulation of phosphatidylinositol 3-kinase. J. Biol. Chem. 274:5333-5338. 36. Kodama, T., Hazeki, K., Hazeki, O., Okada, T., and Ui, M. 1999.

Enhancement of chemotactic peptide-induced activation of phosphoinositide 3-kinase by granulocyte-macrophage colony- stimulating factor and its relation to the cytokine-mediated priming of neutrophil superoxide-anion production. Biochem. J. 337:201- 209.

37. Gomez-Cambronero, J., Huang, C. K., Gomez-Cambronero, T. M., Waterman, W.H., Becker, E. L., and Sha'Afi, R.I. 1992. Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils. Proc. Natl. Acad. Sci. U S A. 89:7551-7555.

38. Durstin, M., Durstin, S., Molski, T.F.P, Becker, E. L., and. Sha'Afi, R.I. 1994. Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase. Proc. Natl. Acad.

Sci. U S A. 91 :3142-3146.

39. McLeish, K.R., Knall, C, Ward, R. A., Gerwins, P., Coxon, P. Y., Klein, J. B., and Johnson. G. L. 1998. Activation of mitogen- activated protein kinase cascades during priming of human neutrophils by TNF-α and GM-CSF. J. Leukoc. Biol. 64:537-545.

40. Suzuki, K., Hino, M., Hato. F., Tatsumi, N., Kitagawa, S. 1999. Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. Blood. 93:341-349.

41. Brizzi, M. F., Aronica, M. G., Rosso, A., Bagnara, G. P., Yarden, Y., Pegoraro, L. 1996. Granulocyte-macrophage colony-stimulating factor stimulates JAK2 signaling pathway and rapidly activates p93fes, STAT1 p91 , and STAT3 p92 in polymorphonuclear leukocytes. J. Biol. Chem. 271 :3562-3567.

42.AI-Shami, A., Mahanna, W., Naccache, PH.1998. Granulocyte- macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Selective activation of Jak2, Stat3, and Statδb. J. Biol. Chem. 273:1058-1063. 43.Aggarwal, B. B., and K. Natarajan. 1996. Tumor necrosis factors: developments during the last decade. Eur. Cytokine. Netw. 7:93. 44. Firestein, G.S., Zvaifler, N.J. 1992. Rheumatoid Arthritis: a disease of disordered immunity. In Inflammation. Basic principles and clinical correlates; Second edition. J.I. GaIMn, I. M. Goldstein; R. Snyderman editors. Raven Press, New York. p959-977. 45. Smith JA. Neutrophils, host defense, and inflammation: a double- edged sword. J. Leukoc. Biol. 1994; 56: 672-686.

46. Elortza, F., Nuhse, T.S., Foster, L.J., Stensballe, A., Peck, S.C., Jensen, O.N. 2003. Proteomic Analysis of Glycosylphosphatidylinositol-anchored Membrane Proteins. MoI. Cell. Proteom. 2:1261-1270. 47.Thompson, H. L., Marshall, C. J., and Saklatvala, J. 1994.

Characterization of two different forms of mitogen-activated protein kinase kinase induced in polymorphonuclear leukocytes following stimulation by N-Formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor. J. Biol. Chem. 269:9486-9492.

48.Alessi, D. R., A. Cuenda, P. Cohen, DT. Dudley, and Saltiel, A.R. 1995. PD098059 is a specific inhibitor of the activation of mitogen- activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270:27489-27494. 49. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J.,

Stradley, D. A., Feeser, W. S., VanDyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. 1998. Identification of a novel inhibitor of mitogen activated protein kinase kinase (MEK). J. Biol. Chem. 273:18623- 18632. 5O. Cuenda, A., Rouse, J., Doza, Y.N., Meier, R., Cohen, P., Gallagher, T.F., Young, P. R., and Lee, J.C. 1995. SB203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 364:229-233. 51. Nagahara, H., Vocero-Akbani, A.M., Snyder, E. L., Ho, A., Latham,

D. G., Lissy, N.A., Becker-Hapak, M., Ezhevsky, S.A., Dowdy, S. F. 1998. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4:1449-1452. 52. Han, H., Fuortes, M., Nathan, C. 2003. Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation. J. Exp. Med. 197:63- 75. 53. El Benna, J., Han, J., Park, J-W., Schmid, E., Ulevitch, R. J., and

Babior, B. M. 1996. Activation of p38 in stimulated human neutrophils: phosphorylation of oxidase component p47phox by p38 and ERK but not by JNK. Arch. Biochem. Biophys. 334:395- 400. 54. Manke, IA, Nguyen, A., Lim, D., Stewart, M.Q., ENa, A.E., Yaffe,

M. B. 2005. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. MoI. Cell. 17:37-48.

55. Park, H-S., Park, J-W. 1998. Conformational changes of leukocyte NADPH oxidase subunit p47phox during activation studied through its intrinsic fluorescence. Biochim. Biophys. Acta. 1387:406-411.

56. Swain, S. D., Helgerson, S. L., Davis, A. R., Nelson, L. K., and Quinn, MT. 1997. Analysis of activation-induced conformational changes in p47phox using tryptophan fluorescence spectroscopy.

J. Biol. Chem. 272:29502-29509. 57. Groemping, Y., Lapouge, K., Smerdon, S.J., Rittinger. K. 2003.

Molecular basis of phosphorylation-induced activation of the

NADPH oxidase. Cell. 113:343-355.

58. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685.

59.Arnett, F.C., Edworthy, S. M., Bloch, D.A, et al. 1988. The

American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31 : 315-324. 60. Dougados, M., Van der Linden, S., Juhlin, R. et al. 1991. The European Spondylarthropathy Study Group preliminary criteria for the classification of spondylarthropathy. Arthritis. Rheum. 34:

1218-1227.

Claims

Claims

1. A polypeptide of 50 amino acids in length or less, comprising the following amino acid sequence:

XPGPQZPGSPL (I) Wherein X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and Z means an amino acid residue S or an amino acid residue T.

2. A polypeptide consisting of the following amino acid sequence: XPGPQZPGSPL (I)

Wherein X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and Z means an amino acid residue S or an amino acid residue T.

3. A polypeptide according to claim 1 , wherein X means an amino acid residue R and Z means an amino acid residue S.

4. A polypeptide according to claim 1 , comprising the following amino acid sequence : ARPGPQSPGSPL (II).

5. A polypeptide consisting of the following amino acid sequence : ARPGPQSPGSPL (II).

6. A polypeptide according to claim 1 of formula :

RKKRRQRRR(α)_nXPGPQZPGSPL (III) wherein :

-n is an integer meaning 0 or 1 ,

- (α) means an amino acid sequence comprising 1 to 10 amino acids. - X means any amino acid residue selected from the group consisting of

D, E, H, K and R ; and - Z means an amino acid residue S or an amino acid residue T.

7. A polypeptide according to claim 1 of formula : RQIKIWFQNRRMKWKK (α)_nXPGPQZPGSPL (IV) wherein :

-n is an integer meaning 0 or 1 ,

- (α) means an amino acid sequence comprising 1 to 10 amino acids.

- X means any amino acid residue selected from the group consisting of D, E, H, K and R ; and - Z means an amino acid residue S or an amino acid residue T.

8. A composition comprising :

(i) a polypeptide according to claim 1-5 as an active ingredient (ii) a mean for delivering the polypeptide according to claim 1-6 in a cell.

9. A composition according to claim 8, wherein the mean for delivering the polypeptide according to claim 1-5, is selected from the group consisting of a liposome, a polymer of lysine, and a polymer of arginine.

10. A pharmaceutical composition comprising a polypeptide according to claims 1-5 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising a composition according to claim 8-9 and a pharmaceutically acceptable carrier.

12. A method for the prevention or the treatment of an inflammatory disorder, comprising a step of administering a polypeptide according to claim 1-5 to a patient.

13. A method for the prevention or the treatment of an inflammatory disorder, comprising a step of administering a composition according to claim 8.

14. A method for the prevention or the treatment of an inflammatory disorder, comprising a step of administering a pharmaceutical composition according to claim 10.

15. A method according to claim 12-14 wherein said inflammatory disorder is selected from the group consisting of psoriasis and dermatitis, Crohn's disease, ulcerative colitis, ischemic reperfusion; adult respiratory distress syndrome; meningitis; encaphalitis; uveitis; rheumatoid arthritis, Sjogren's syndrome, vasculitis; diseases involving leukocyte diapedesis; multiple organ injury syndrome secondary to septicaemia or trauma; alcoholic hepatitis; bacterial pneumonia; hypovolemic shock; glomenulanephritis; multiple sclerosis; Type I diabetes melitis; acute and delayed hypersensitivity, graft vs. host disease; transplant rejection; reperfusion injury; endotoxic shock; disease states due to leukocyte dyscrasia and metastasis; asthma; pulmonary oxygen toxicity; pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, and cystic librosis, stroke-induced brain cell death, ankylosing spondylitis, fibromyalgia, systemic lupus erythematosus, scleroderma, and systemic sclerosis.

16. A method according to claim 15 wherein said inflammatory disorder is preferably selected from the group consisting of rheumatoid arthritis, Crohn's disease or ankylosing spondylitis.

17. An antibody directed against a polypeptide of formula : QARPGPQS(phospho)PGSPLEEER (V), Wherein "S(phospho)" means a phosphorylated serine amino acid residue.

18. A method for detecting an inflammatory disorder in an individual, comprising the following steps :

(a) incubating a biological sample from said individual with an antibody according to claim 16, and

(b) detecting the binding of said antibody with the P47phox protein of sequence SEQ ID N°1 , wherein the binding of said antibody is indicative of the presence of an inflammatory disorder in said individual.