DE102008014880A1 - Use of a polypeptide with the activity of repulsive guidance molecule A as an antiinflammatory agent - Google Patents

Abstract

Polypeptide (I) with the activity of repulsive guidance molecule A is used as an antiinflammatory agent. Independent claims are also included for: (1) nucleic acid coding for (I); (2) expression vector and host containing the nucleic acid. ACTIVITY : Antiinflammatory. MECHANISM OF ACTION : Neutrophil migration inhibitor.

Description

The The present invention relates to an anti-inflammatory polypeptide. a nucleic acid molecule responsible for a such polypeptide encodes an expression vector containing such Nucleinsäuremolekül, a host, the the nucleic acid molecule or the expression vector comprising a pharmaceutical composition comprising the polypeptide, Having nucleic acid molecule or expression vector as well as a method of treating an inflammatory Illness.

A Inflammation is a characteristic response of biological Tissue on an external or internally triggered Stimulus with the function, the stimulating agent and its Eliminate consequences. During the inflammatory reaction it comes to the migration of inflammatory cells, for example of leukocytes, in place of infection. An excessive migration However, inflammatory cells in organs can be destroyed of the affected tissue, the uncontrolled inflammatory response and ultimately to autoimmunity and an associated permanent Cause inflammatory reaction. You already know a variety of human diseases characterized by excessive inflammatory responses are.

excessive Inflammatory reactions are currently using anti-inflammatory Agents, such as corticosteroids, non-steroidal anti-inflammatory Treated substances (NSAIDs) and cytokine antagonists. The usage however, such anti-inflammatory agents are common accompanied by significant side effects, their clinical efficacy limit.

In front In this background, it is an object of the present invention to provide new anti-inflammatory agent, the causative agent engages in the inflammatory process and the for described in the prior art known anti-inflammatory agents If possible, do not have side effects.

The The object underlying the invention is achieved by the provision a (poly) peptide, which (a) the biological activity of the "Repulsive Guidance Molecule A" (RGM-A), and / or (b) from a monoclonal / polyclonal α-RGM-A antibody is specifically bound.

The Inventors have surprisingly recognized that the "repulsive Guidance Molecule A "(RGM-A) causally in the Engage and inhibit the inflammatory process. This finding was therefore surprising since RGM-A so far another function was attributed.

Human RGM-A was first used by Monnier et al. (2002), "RGM is a repulsive guidance molecule for retinal axons" Nature 419 (6905), 392-5 , described. It has 485 amino acids on it. The amino acid sequence is shown in the NCBI database (GeneBank) under accession number AAH15886 and in the attached sequence listing under SEQ ID NO: 1. The coding sequence of RGM-A in its human variant is shown in the NCBI database (GeneBank) under accession number BC015886 and under SEQ ID NO: 2 in the attached sequence listing.

Schwab, JM et al. (2005), "Central nervous system injury-induced repulsive guidance molecule expression in the adult human brain." Arch. Neurol. 62, 1561-8 describe, for example, that RGM-A is a neuronal pathway molecule that is expressed in the central nervous system and is a chemical attractant or, in neuronal development, a repulsive chemical factor. The signal of RGM-A on the nervous system is thereby mediated by the neogenin receptor and partly by the DCC receptor; see. Hata, K. et. al. (2006), "RGMa inhibition promotes axonal growth and recovery after spinal cord injury", J. Cell. Biol. 173, 47-58 " and Matsunaga, E. et al. (2004), "RGM and its receptor neogenin regulate neuronal survival", Nat. Cell. Biol. 6, 749-55 , The DCC receptor is not expressed on leukocytes, but a subtype of the neogenin receptor, namely the UNC5b receptor; see. Ly, NP et al. (2005), "Netrin-1 inhibits leukocyte migration in vitro and in vivo", Proc. Natl. Acad. Sci. USA 102, 14729-34 ,

A Expression and / or activity of RGM-A outside of the central nervous system has not been described so far.

For a professional can easily determine whether the polypeptide according to the invention the activity of RGM-A. For example, in a transmigration assay, described in more detail in the embodiments will determine if the migration of neutrophilic granulocytes on chemotactic stimuli by the invention Peptide is inhibited. If a detected inhibition points the polypeptide has the biological activity of RGM-A.

The required activity of the polypeptide according to the invention can be further determined by demonstrating that the polypeptide can bind specifically to a monoclonal and / or polyclonal antibody directed against RGM-A. Monoclonal and polyclonal α-RGM-A antibodies are known in the art, for example the following:
R & D Systems, 614 McKinley Place NE, Minneapolis 55413, USA: α-chicken RGM-A anti body, affinity purified polyclonal antibody (catalog number AF2010); α-chicken RGM-A antibody (clone 292012), monoclonal antibody (catalog number MAB2010); biontinylated α-chicken RGM-A antibody, affinity-purified polyclonal antibody (catalog number: BAF2010); biotinylated α-human RGM-A antibody, affinity purified polyclonal antibody (catalog number: BAF2459); α-human / mouse / chicken RGM-A antibody (clone 275407), monoclonal antibody (catalog number MAB2458); α-mouse RGM-A antibody, affinity purified polyclonal antibody (catalog number AF2458); biotinylated α-mouse RGM-A antibody, affinity-purified polyclonal antibody (catalog number BAF2458).

company Santa Cruz Biotechnology, Inc. 2145 Delaware Avenue, Santa Cruz, California 95060, USA: α-human / mouse / rabbit RGM-A antibody, affinity-purified polyclonal antibody against the C-terminus (catalog number sc-46481); α-human / mouse / rabbit-RGM A antibodies, affinity-purified polyclonal antibody against the internal section (catalog number sc-46482); α-human / mouse / rabbit-RGM A antibodies, affinity-purified polyclonal antibody against amino acids 291 to 365 (catalog number sc-67052); α-human / mouse / rabbit-RGM A antibodies, affinity purified polyclonal antibody against an area near the C-terminus (catalog number sc-46484).

company Axxora Marie-Curie-Straße 8, 79539 Lörrach, Germany: α-human RGM-A antibody, affinity purified polyclonal antibody (catalog number ALX-210-937-C100).

company Abcam, 332 Cambridge Science Park, Cambridge CB4 0FW, United Kingdom: α-human / mouse / rat RGM-A antibody, affinity-purified polyclonal antibody (Catalog number from26287).

As The inventors have found that all of the above bind Antibody to the polypeptide of the invention in a specific way.

The Binding of the antibody is specific, though these too under stringent conditions. Stringent conditions can for example, be created by a corresponding binding buffer, having, for example, the following composition: 1% normal goat serum, 0.1% bovine serum albumin (BSA) in physiological saline (pH 7.4%).

According to the invention understood as an association under an anti-inflammatory agent, those for the treatment and / or prophylaxis of inflammation be used in a living being, like a human, and that Reduce the extent of the inflammatory reaction or can inhibit.

The The object underlying the invention is hereby completely solved.

To a preferred embodiment, the inventive Polypeptide to an amino acid sequence, at least to 80%, preferably 85%, more preferably 90%, more preferably 95%, most preferably 99% identical to the amino acid sequence SEQ ID NO: 1 on the attached sequence listing.

These Measure has the advantage that for the production of the inventive Polypeptides provided alternative primary structures which may be easier to synthesize than the amino acid sequence SEQ ID NO: 1. The amino acid sequence SEQ ID NO. 1 gives the Primary structure of human RGM-A again. To a polypeptide with the biological activity of RGM-A and thus one Anti-inflammatory agent according to the invention it is not mandatory to obtain a polypeptide to provide an amino acid sequence which 100% identical to the amino acid sequence SEQ ID NO. 1. It is sufficient, if sufficiently high identity given, with possibly moderate activity losses are tolerable. The specified identities relate to the entire amino acid sequence SEQ ID NO. 1 or to a Section thereof with ≥ 10 amino acids.

Of the Degree of homology between the invention Polypeptide and RGM-A or SEQ ID NO: 1 can be easily determine by means of methods known in the art, for example BLAST analysis or using the MegAlign module of the Lasergene program by DNAStar Inc.

One Another object of the present invention relates to a polypeptide, the amino acid sequence SEQ ID NO. 1 from the attached Sequence listing has.

With This measure provides a polypeptide which the human variant of RGM-A corresponds and therefore in particular especially suitable for use in humans as anti-inflammatory agent is.

According to a preferred development, the polypeptide according to the invention has an amino acid sequence which corresponds to SEQ ID NO. 1, in which at least (a) one polar against another polar amino acid, and / or (b) one non-polar one against another non-polar one. polar amino acid, and / or (c) one acidic against another acidic amino acid, and / or (d) one basic versus another is exchanged.

These Measure has the advantage of providing a polypeptide becomes, which differs from SEQ ID No. 1 amino acid sequence has, however, the inventive anti-inflammatory property includes. Thus, it is known in the art that in a protein one amino acid against another amino acid with similar ones Biochemical properties can be exchanged without As a result, the biological activity changes significantly. Among the interchangeable polar amino acids include glycine, tyrosine, tryptophan, asparagine, glutamine, Serine, treonin and cysteine. To the non-polar amino acids include alanine, valine, leucine, isoleucine, phenylalanine, Metionin and proline. The acidic amino acids include aspartic acid and glutamic acid. To the basic amino acids include histidine, arginine and lysine. These amino acids have similar chemical properties and behavior therefore comparable in a protein, so that the folding and the activity are largely identical.

there it is preferred if in the inventive Polypeptide biological activity of RGM-A inhibition Migration of neutrophilic granulocytes to chemotactic stimuli represents.

These A measure has the advantage of providing such a polypeptide which has the property of RGM-A, which according to findings the inventor responsible for its anti-inflammatory effect is. Furthermore, this property can be easily by means of of a transmigration assay and therefore can be used to identify and characterizing the polypeptide of the invention become.

One Another object of the present invention relates to a nucleic acid molecule, which is preferably DNA or cDNA, and for the previously described Coded polypeptide according to the invention.

The Nucleic acid molecule has the coding sequence for the polypeptide according to the invention and therefore can serve as a template for the preparation of the latter with recombinant DNA technology. Another Advantage of a nucleic acid molecule is that it is much more stable than a protein and therefore close to store indefinitely.

there it is preferred if the nucleic acid molecule the nucleotide sequence SEQ ID NO: 2 from the enclosed sequence listing or a variant thereof degenerated due to the genetic code having.

SEQ ID No. 2 gives the nucleotide sequence of the human variant of RGM-A again and therefore encodes a preferred invention Polypeptide. It is understood that with the invention, such Nucleic acid molecule is included in the opposite nucleotide sequence SEQ ID NO: 2 exchanged individual nucleotides were that affected codon, however, still for the same amino acid encodes as the corresponding codon SEQ ID NO: 2. This is the degeneration of the genetic code exploited, according to the different codons for the same Code amino acid.

One Another object of the present invention relates to an expression vector, the nucleic acid molecule of the invention having.

These Measure has the advantage of providing an item is, with the polypeptide of the invention Practical can be made in unlimited quantities. It come both prokaryotic and eukaryotic expression vectors in question, if necessary, regulatory nucleotide sections such as promoters and enhancers. Furthermore, the inventive Expression vector restriction endonuclease sites, Coding sequences for another protein, such as. glutamine S-transferase (GST) to a fusion protein, or other amino acids, eg. As histidines, for the preparation of a Histidine tagged RGM-A protein, etc.

One Another object of the present invention relates to a host, the expression vector according to the invention or the nucleic acid molecule of the invention having.

at the host is preferably a prokaryotic cell or a bacterial cell, for example Escherichia coli. through such a host can easily be used in cell culture the polypeptide of the invention in large Quantities are produced. In question is also one eukaryotic cell, such as an insect cell, a non-human mammal etc. In the case of a non-human mammal becomes a model system provided with scientific research on the mode of action of RGM-A during the inflammatory process can be examined.

One Another object of the present invention relates to a pharmaceutical Composition containing the polypeptide of the invention and / or the nucleic acid molecule of the invention and / or the expression vector according to the invention and a pharmaceutically acceptable carrier and optionally has further active ingredients.

This article is already a ready-made drug provided with the inflammatory processes can be prevented or inhibited. Pharmaceutically acceptable carriers are extensively described in the art, for example, in U.S. Pat Rowe et al. (2006), Handbook of Pharmaceutical Excepients, Pharmaceutical Press , The content of this publication is incorporated herein by reference. Further active ingredients may be provided, preferably further antiinflammatory substances mentioned in the beginning.

One Another object of the present invention relates to a method for the treatment of an inflammatory disease in a Living beings with the following steps: (a) Provision of a anti-inflammatory agent, (b) administration of the anti-inflammatory agent Agent in a living being, (c) if necessary repetition of steps (a) and (b), wherein the anti-inflammatory agent according to the invention (Poly) peptide is provided.

The The invention will now be described in more detail with reference to exemplary embodiments explains what makes up more features and benefits result. Reference is made to the attached figures, where the following is shown:

• A) Das synthetische Peptid formyl-Met-Leu-Phe (fMLP) (10 ng/ml) induziert die Chemotaxis von neutrophilen Granulocyten (PMN) durch eine Monoschicht von humanen Coloncarcinomzellen (CaCo-Zellen) (Costar, 3 μm Porengröße). 1 × 10⁶ PMN wurden in das apikale Kompartiment platziert und nach 60 Minuten wurde die Transmigration von PMN gemessen.
• B) RGM-A reduziert die PMN-Transmigration auf dosisabhängige Art und Weise. PMN wurden mit zunehmenden Dosen von RGM-A präinkubiert und dann in das apikale Kompartiment einer Transmigrationskammer platziert. Die Chemotaxis wurde durch fMLP (10 ng/ml, 60 Minuten) induziert und in dem basalen Kompartiment wurde die Myeloperoxidaseaktivität (MPO) gemessen.
• C) RGM-A inhibiert die fMLP-induzierte PMN-Migration, wenn RGM-A entweder in dem apikalen Kompartiment oder dem basalen Kompartiment der Transmigrationskammer vorhanden ist. PNM wurden in die obere Kammer platziert und mit 10 nm fMLP wurde die Migration in die untere Kammer induziert. In Gegenwart und Abwesenheit von RGM-A (500 ng/ml) wurde die MPO-Aktivität in dem basalen Kompartiment in der oberen, der unteren oder in beiden Kammern gemessen.

1 illustrates the inhibition of migration of neutrophil granulocytes to chemotactic stimuli by RGM-A in vitro.

• A) The synthetic peptide formyl-Met-Leu-Phe (fMLP) (10 ng / ml) induces the chemotaxis of neutrophilic granulocytes (PMN) through a monolayer of human colon carcinoma cells (CaCo cells) (Costar, 3 μm pore size). 1 x 10 ⁶ PMN was placed in the apical compartment and after 60 minutes PMN transmigration was measured.
• B) RGM-A reduces PMN transmigration in a dose-dependent manner. PMN were preincubated with increasing doses of RGM-A and then placed in the apical compartment of a transmigration chamber. Chemotaxis was induced by fMLP (10 ng / ml, 60 minutes) and myeloperoxidase activity (MPO) was measured in the basal compartment.
• C) RGM-A inhibits fMLP-induced PMN migration when RGM-A is present in either the apical compartment or the basal compartment of the transmigration chamber. PNM were placed in the upper chamber and with 10 nm fMLP, migration into the lower chamber was induced. In the presence and absence of RGM-A (500 ng / ml), MPO activity in the basal compartment was measured in the upper, lower or both chambers.

All Data represents mean ± SEM, n = 9 per condition, * P <0.05.

• A) Die MPO-Aktivität in dem basolateralen Kompartiment wurde gemessen, nachdem ein zuvor die PMN mit α-RGM-A-Antikörper oder hitzeinaktiviertes RGM-A (HI-RGM-A, 500 ng/ml, 95°C, 30 Minuten) präinkubiert wurden. Die Chemotaxis wurde erneut mit fMLP (10 ng/ml, 60 Minuten) induziert.
• B) Die Behandlung von PMN mit α-UNC5b-Antikörper hebt dosisabhängig den inhibitorischen Effekt von RGM-A auf die PMN-Migration auf. Die Behandlung von PMN mit einem IgG-Isotyp-Kontroll- oder α-BMP2/4-Rezeptor-Antikörper hingegen hebt den inhibitorischen Effekt von RGM-A auf die PMN-Migration nicht auf.

2 demonstrates that inhibition of neutrophil granulocyte migration by RGM-A is mediated via a UNC5b-dependent mechanism.

• A) The MPO activity in the basolateral compartment was measured after one previously had the PMN with α-RGM-A antibody or heat-inactivated RGM-A (HI-RGM-A, 500 ng / ml, 95 ° C, 30 minutes). were preincubated. Chemotaxis was induced again with fMLP (10 ng / ml, 60 minutes).
• B) Treatment of PMN with α-UNC5b antibody dose-dependently abrogates the inhibitory effect of RGM-A on PMN migration. Treatment of PMN with an IgG isotype control or α-BMP2 / 4 receptor antibody, on the other hand, does not abrogate the inhibitory effect of RGM-A on PMN migration.

All Data represents mean ± SEM, n = 9 per condition, * P <0.05.

• A) Die RGM-A-mRNA-Expression in Mausgeweben wurde durch eine quantitative Echtzeit RT-PCR quantifiziert. Jede Probe wurde in Dreifachansätzen analysiert und die Menge von Netrin-1 wurde im Vergleich zur relativen Expression im Gehirn berechnet.
• B) RGM-A inhibiert die Leukozytenrekrutierung in vivo in einem Modell der durch Zymosan A (ZyA) induzierten Peritonitis bei der Maus. Mäusen wurde i. p. ZyA (1 ml, 1 mg/ml) injiziert, um Peritonitis zu induzieren. Die Tiere wurden entweder mit Vehikel (1% BSA in PBS) oder RGM- A (1 μg) i. v. behandelt und nach 4,8 und 24 Stunden wurde eine peritoneale Lavage durchgeführt.
• C) Nach der ZyA-induzierten Peritonitis wurde in der peritonealen Lavage die MPO-Aktivität gemessen.
• D) Cytospin-Proben der peritonealen Lavage in VT-Mäusen, entweder behandelt mit Vehikel oder RGM-A, und zwar nach 4,8 und 24 Stunden.

3 demonstrates that the migration of neutrophilic granulocytes to chemotactic stimuli by RGM-A is also inhibited in vivo.

• A) The RGM-A mRNA expression in mouse tissues was quantitated by quantitative real-time RT-PCR. Each sample was analyzed in triplicate and the amount of netrin-1 was calculated in comparison to the relative expression in the brain.
• B) RGM-A inhibits leukocyte recruitment in vivo in a model of zymosan A (ZyA) -induced mouse peritonitis. Mice were injected ip with ZyA (1 ml, 1 mg / ml) to induce peritonitis. The animals were treated either with vehicle (1% BSA in PBS) or RGM-A (1 μg) iv and after 4,8 and 24 hours peritoneal lavage was performed.
• C) After the ZyA-induced peritonitis, MPO activity was measured in the peritoneal lavage.
• D) Cytospin samples of peritoneal lavage in VT mice, either treated with vehicle or RGM-A, at 4.8 and 24 hours.

All Data represents mean ± SEM, n = 9 per condition, * P <0.05.

embodiments

1. Material and methods

1.1 Analysis of transcription and protein expression

Using a real-time PCR RT-PCR (iCycler, Bio-Rad Laboratories Inc.), a semi-quantitative analysis was performed to examine the levels of RGM-A expression in mouse tissue. The tissue was and RNA isolated using Trizol according to standard protocol. The primer sets contained 10 pM of the sense primer 5'-CAG CAA GCT CAC CAT CAT CT-3 '(SEQ ID NO: 3) and the antisense primer 5'-CCC GAC ACC TTC TCT GTG AT-3' (SEQ ID NO: 4) for the analysis of the mouse tissue. The primer set was amplified by performing an increasing number of cycles of 95 ° C for 1 minute, 64 ° C for 2 minutes, 72 ° C for 1 minute, and a final extension at 72 ° C for 7 minutes. The samples were monitored by β-actin using appropriate PCR primers.

For Western blot analysis homogenized the animal samples for protein levels normalized and under non-reducing conditions separated by means of SDS-containing polyacrylamide gels. In the Western blot analysis were monoclonal mouse α-netrin-1 antibodies (Santa Cruz Biotechnology) for mouse RGM-A analysis and α-rabbit peroxidase used coupled secondary antibodies. The actin was using rabbit α-actin antibody (Santa Cruz Biotechnology) stained. The blots were washed and the respective specific peroxidase-coupled Secondary antibodies were added. The gangs The washed blots were enhanced by chemiluminescence (Amersham Pharmacia Biotech).

1.2 Immunohistochemistry

The Mouse tissue was recovered after further processing frozen in a suitable medium. 5 μm thick sections were placed on microscope slides, air-dried, in methanol and then fixed in 4% acetone. The air-dried Lung sections and cells were washed three times in PBS and washed with 5% BSA (Sigma Chemicals) blocked. The primary antibodies used included u. a. α-human RGM-A rabbit antibody (Santa Cruz Biotechnology; sc-46482). Experimental Negative Controls were prepared in PBS / 2% BSA with rabbit IgG made by the inventors , Goat IgG (Santa Cruz Biotechnology) and Mouse IgG (Santa Cruz Biotechnology) incubated. The secondary antibodies included u. a. FITC-conjugated α-rabbit donkey antibodies against Hoechst 33342 marker (blue) (Invitrogen) to the cell nuclei to stain.

1.3 Isolation of human neutrophils Granulocytes and transmigration assays

These Studies were approved by the local ethics committee. The Agreement after comprehensive information the participants were informed according to the Declaration of Helsinki obtained. Neutrophil granulocytes (PMNs) were isolated from whole blood, that of human volunteers obtained by puncture of the vein and anticoagulated with citric acid / dextrose has been. The resulting cell population contained more than 97% PMNs, as determined by microscopic evaluation. The PMNs were within examined 2 hours after their isolation.

PMNs were with increasing concentrations of RGM-A, with 500 being ng / ml and with bovine serum albumin (BSA) as the control protein incubated at the same concentrations. CaCo cells were on permeable inserts (3 μm pore size) was grown to confluency and before use the resistance measured (7-10 days after the Plating). The monolayers were washed and for further investigations in Hanks Balanced Salt Buffer (HBSS +) placed as previously described.

1 x 10 ⁷ PMNs were then placed on the apical side of the monolayers and incubated for 60 minutes. 10 nM fMLP, which simulates the activity of bacterial peptides, was added to the basolateral compost and transmigration was assayed by a myeloperoxidase (MPO) assay as described previously in US Pat Lawrence, DW et al. (2003), "Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia", J. Exp. Med. 198, 999-1010 to determine the basolateral PMN content. In a series of experiments, PMNs were incubated with specific α-UNC5b, an α-neogenin and an α-BMP2 / 4 receptor antibody at decreasing concentrations to study the influence on PMN transmigration. PMNs were then incubated with 500 ng / ml netrin-1 and examined in the Transwell assay for 60 minutes. The resulting PMN transmigration was determined using the MPO assay.

1.4 Peritonitism Model

All Animal experiments were in accordance with the regulations of the regional council Tübingen and the local ethics committee. The mice were obtained from Charles River Laboratory (8 to 12 week old male / female C57BL / 6 mice, n = 6 per group). The animals were either i. v. Vehicle (saline + 0.2% BSA) or a 1 μg recombinant mouse RGM-A (150 μl Total volume). Next were the mice i. p. 1 ml zymosan a (1 mg / ml) was injected and incubated for 4, 8 and 24 hours.

The recruited leukocytes were harvested at the time indicated by peritoneal lavage with calcium- and magnesium-free and ice-cold PBS buffer unfractionated at 10 U / ml Heparin contained. The cells were counted, labeled with specific antibodies and fixed for FACS analysis. The collected cells were washed, resuspended in 2 ml of HBSS + and counted. Cytospin samples were prepared and the cells were stained with Diff-Quick to distinguish the subpopulations of leukocytes. All detergents used were free of endotoxin.

2 results

2.1 RGM-A modulates the migration of neutrophilic granulocytes in vitro.

To evaluate whether RGM-A affects the migration of inflammatory cells, it was first tested whether a model of fMLP-induced chemotaxis is suitable for evaluating the migration of neutrophilic granulocytes (PMN) in response to chemotactic molecules. It was found that fMLP significantly induces the migration of PMNs through a monolayer of CaCo epithelial cells placed on semipermeable Transwell inserts; see. 1A ,

This fMLP-induced migration of PMNs through a layer of epithelial cells was dose-dependently reduced by RGM-A; see. 1B , 500 ng / ml RGM-A reduced the migration of PMNs in response to fMLP by 44% (± 6%). This reduction in PMN transmigration was not observed when the PMNs were exposed to a non-specific protein at the same concentrations as bovine serum albumin (BSA) (data not shown).

To further demonstrate the potential of RGM-A to reduce PMN migration, PMNs on the apical or basolateral side of the layer of epithelial cells were exposed to RGM-A. RGM-A was added selectively to either side of the transmigration chamber. Apical exposure to RGM-A significantly reduced PMN migration compared to the situation where RGM-A was added to the basolateral compartment; see. 2C ,

2.2 The RGM-A inactivation leads to a weakened effect

Around To evaluate the functional role of RGM-A on PMNs was first the possibility of inactivating the biological effect from RGM-A to PMNs. For this purpose was prior to exposure to PMNs, an antibody directed against RGM-A added to the RGM-A protein. In the subsequent transmigration assay it was found that the antibody was the one previously observed Function of RGM-A attenuated PMN transmigration and ultimately to a complete neutralization of RGM-A function resulted.

Next, the tertiary structure of the RGM-A protein was heat inactivated and the inactivated protein was re-exposed to PMNs. It was found that the previously observed effect on PMN migration was not observed when the PMNs were exposed to heat-inactivated RGM-A; see. 2 B ,

2.3 RGM-A mediates its effect PMNs through the UNC5b receptor

In the next step, the signaling mechanism underlying the anti-migratory potential of RGM-A was investigated. In the central nervous system, RGM-A presumably mediates its function via the neogenin receptor; a subtype of this receptor, UNC5b, has recently been identified on PMNs. Therefore, the PMNs were first exposed to an α-UNC5b antibody before exposure of these PMNs to RGM-A. The transmigration assay found that with increasing antibody levels, the observed RGM-A function was completely attenuated; see. 2 B ,

Around to exclude non-specific effects of the antibody, the same experimental protocol was performed with the isotype control antibody and the inhibitory effect of RGM-A on PMN migration again observed.

As another receptor stimulated by RGM-A, the BMP2 / 4 receptor is discussed. To rule out the possibility that this receptor influences the observed antimigrative effect of RGM-A, an antibody against this receptor was also used before the PMNs were exposed to RGM-A. No effect of BMP 2/4 antibody on the effect of RGM-A on PMN migration was observed; see. 2 B ,

2.4 RGM-A will be outside the expressed central nervous system and has anti-inflammatory potential

So far, the expression of RGM-A outside the central nervous system has not been studied. Therefore, a variety of organs were first screened for the presence of RGM-A mRNA. It was found that RGM-A is expressed in a variety of tissues outside the central nervous system, such as the heart, spleen, lung, and liver, and to a lesser extent, in the intestine and kidney; see. 3A ,

Next, a model of zymosan A induced peritonitis was used to evaluate a potential effect of RGM-A on inflammatory processes in vivo. Animals were injected iv with either vehicle (NaCl + 0.2% BSA) or 1 μg RGM-A. After 4, 8 and 24 hours a peritoneal lavage was performed. It was found that the number of inflammatory cells after 4 hours was significantly reduced by the application of RGM-A; see. 3B , The reduction was still observable after 8 hours. After 24 hours, however, no reduction was observed.

This result was reflected by the measurement of MPO in the peritoneal lavage. Thus, it was found that the MPO activity was significantly reduced after 4 hours; see. 3C ,

To visualize the reduction of the inflammatory cells, a peritoneal lavage cytospin was performed and the same result was obtained as in the previously described experiment; see. 3D ,

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• Monnier et al. (2002), "RGM is a repulsive guidance molecule for retinal axons" Nature 419 (6905), 392-5 [0007]
• Schwab, JM et al. (2005), "Central nervous system injury-induced repulsive guidance molecule expression in the adult human brain." Arch. Neurol. 62, 1561-8 [0008]
• - Hata, K. et. al. (2006), "RGMa inhibition promotes axonal growth and recovery after spinal cord injury", J. Cell. Biol. 173, 47-58 " [0008]
• Matsunaga, E. et al. (2004), "RGM and its receptor neogenin regulate neuronal survival", Nat. Cell. Biol. 6, 749-55 [0008]
• Ly, NP et al. (2005), "Netrin-1 inhibits leukocyte migration in vitro and in vivo", Proc. Natl. Acad. Sci. USA 102, 14729-34 [0008]
• Rowe et al. (2006), Handbook of Pharmaceutical Excepients, Pharmaceutical Press [0037]
• Lawrence, DW et al. (2003), "Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia", J. Exp. Med. 198, 999-1010 [0051]

Claims (13)

(Poly) peptide, the (a) the biological activity of the "Repulsive Guidance Molecule A" (RGM-A), and or (b) from a monoclonal / polyclonal α-RGM-A antibody is specifically bound for use as anti-inflammatory Agent. The (poly) peptide of claim 1 which has an amino acid sequence which is at least 80%, preferably 85%, more preferred at 90%, more preferably at 95%, most preferably at 99% is identical to the amino acid sequence SEQ ID NO the attached sequence listing. The (poly) peptide of claim 1 which has the amino acid sequence SEQ ID NO: 1 from the enclosed sequence listing. The (poly) peptide of claim 1 or 2 which has an amino acid sequence having the SEQ ID NO. 1, in the at least (A) a polar against another polar amino acid, and / or (B) one nonpolar against another nonpolar amino acid, and or (c) one acidic against another acidic amino acid, and / or (D) one basic against another basic amino acid exchanged is. (Poly) peptide according to one of the preceding claims, characterized in that the biological activity the inhibition of migration of neutrophilic granulocytes to chemotactic Stimuli is. Nucleic acid molecule suitable for the (poly) peptide according to one of claims 1 to 5 encoded. Nucleic acid molecule according to claim 6, which is DNA. Nucleic acid molecule containing the nucleotide sequence SEQ ID NO: 2 from the enclosed sequence listing or a due of the genetic code degenerate variant thereof. Expression vector containing the nucleic acid molecule according to one of claims 6 to 8. A host expressing the expression vector of claim 9 and / or the nucleic acid molecule according to any one of Claims 6 to 8. The host of claim 10, which is a prokaryotic cell is. Pharmaceutical composition containing the (poly) peptide according to any one of claims 1 to 5, and / or the nucleic acid molecule according to any one of claims 6 to 8 and / or the expression vector according to Claim 9 and a pharmaceutically acceptable carrier and optionally further active ingredients. Method of treating an inflammatory Disease in a living being with the following steps: (A) Provision of an anti-inflammatory agent, (B) Administration of the anti-inflammatory agent into a living being, (C) if necessary, repetition of steps (a) and (b), characterized, that as anti-inflammatory agent, the (poly) peptide according to a of claims 1 to 5 is provided.