EP0354934A1

EP0354934A1 - Neutrophil-activating polypeptide, process for its manufacture and its use as a drug and diagnosticum

Info

Publication number: EP0354934A1
Application number: EP88909527A
Authority: EP
Inventors: Enno Christophers; Jens-Michael SCHRÖDER
Original assignee: Ferring Arzneimittel GmbH
Current assignee: Ferring Arzneimittel GmbH
Priority date: 1987-11-06
Filing date: 1988-11-05
Publication date: 1990-02-21
Also published as: DE3737703A1; WO1989004325A1; JPH02502097A

Abstract

Un polypeptide activateur de neutrophiles isolé de cellules mononucléaires humaines stimulées a un poids moléculaire de 10 000 daltons et présente une seule crête lorsqu'il est soumis à un procédé de chromatographie de fluides sous haute pression à phase inversée. Selon le procédé de production de ce polypeptide, on stimule des cellules mononucléaires humaines avec des lipopolysaccharides, de la phythémagglutinine et/ou des phorbolesters, on sépare le résidu par chromatographie colloïdale, puis on procède à des séparations ultérieures par chromatographie de fluides sous haute pression à échange de cations, par chromatographie de fluides sous haute pression à exclusion et par chromatographie de fluides sous haute pression à phase inversée. On peut utiliser ce polypeptide localement ou systémiquement afin d'augmenter la défense immunitaire cellulaire associée aux phagocytes, ce qui le rend utile comme agent thérapeutique. Il peut en outre être utilisé comme agent de diagnostic et comme réactif pour la production d'anticorps monoclonaux.A neutrophil activator polypeptide isolated from stimulated human mononuclear cells has a molecular weight of 10,000 daltons and has a single peak when subjected to a reverse phase high pressure fluid chromatography process. According to the process for producing this polypeptide, human mononuclear cells are stimulated with lipopolysaccharides, phythemagglutinin and / or phorbolesters, the residue is separated by colloidal chromatography, then subsequent separations are carried out by high pressure fluid chromatography with cation exchange, by exclusion high pressure fluid chromatography and by reverse phase high pressure fluid chromatography. This polypeptide can be used locally or systemically to increase the cellular immune defense associated with phagocytes, which makes it useful as a therapeutic agent. It can also be used as a diagnostic agent and as a reagent for the production of monoclonal antibodies.

Description

Neutrophil activating polypeptide, process for its preparation and its use as a medicament and diagnostic agent

The invention relates to a neutrophil activating polypeptide which has been isolated from human mononuclear cells. This polypeptide is active for human neutrophil granulocytes and is therefore suitable as a medicament, in particular for increasing the cellular phagocyte-associated defense against inflammation and for stimulating granulation tissue and wound healing. In addition, it is suitable as a diagnostic agent for testing the reactivity of the neutrophilic polymorphonuclear leukocytes in vivo or in vitro and as a reagent for the production of monoclonal antibodies using as antigen.

A polymorphonuclear leukocyte (PMNL) activating factor released by human monocytes is known. In addition to chemotaxis (T.Tono-oka et al., 1980, Immunology 39: 607), this factor appears to be related to PMNL degranulation (Klemper et al., 1978, J. Clin. Invest. 61: 1330) and the oxygen-dependent metabolism activate (Klempner et al., 1979, J. Clin. Invest. 64: 996). Subsequent studies have shown that partially purified interleukin-1 preparations show these PMNL-stimulating activities (Sander et al., 1984, J. Immunol. 132: 828; Smith et al., 1985, J. Leucocyte Biology 37: 746), from which it was concluded that IL-1 is a PMNL activating cytokine.

IL-1 denotes a series of biochemically closely related cytokines that are produced by different cell types and includes the lymphocyte activating factor (LAF), endogenous pyrogen (EP), endogenous leukocyte mediator (LEM) or the mononuclear one Cell factor (MCF). These factors are believed to produce numerous biological effects such as fever, fibroblast proliferation, production of acute phase proteins, stimulation of muscle protein catabolism, and neutrophil chemotaxis (Dinarello, CA; 1896, The Year in Immunology 2 : 68).

IL-1 activity has been described in various biochemical forms, i.e. H. 17 kD species with IP 6, 8, 5, 4 and 5, 2, as well as forms with high molecular weights, with similar IP values. To date, however, it is not clear whether the biological properties of IL-1 can be attributed to a single molecular species of IL-1 or not.

In the context of the present invention, a neutrophil-activating peptide (hereinafter also referred to as MONAP) has been found which differs from IL-1.

The invention therefore relates to a new, physiologically active polypeptide with a selective chemotactic effect on neutrophilic, polymorphonuclear leukocytes (MONAP). It also relates to the process for its production, to medicinal products containing it and to its use.

The neutrophil-activating polypeptide according to the invention is isolated from human mononuclear cells stimulated with lipopolysaccharides, phythaemagglutin and / or phorbol esters. It is homogeneous and has a molecular weight of 10 kD as determined by a single band in sodium dodecyl sulfate-polyamide gel electrophoresis and by a single peak in high pressure liquid chromatography (HPLC) (exclusion chromatography). A single peak was also achieved in reversed phase high pressure liquid chromatography (RP-HPLC).

The polypeptide according to the invention can be defined chemically and physically as follows: a) it is a polypeptide; b) molecular weight determined by SDS electrophoresis: 10 kD; c) molecular weight determined by exclusion chromatography (TSK-2000-HPLC), likewise 10 kD; d) it binds to anion exchangers at pH 8; e) binds to "Reversed-Phase (RP-18)"column; f) it is stable under the following conditions:

1) 10 min / 60 ° C

2) 5 min / 100 ° C

3) multiple freezes and thaws

4) mild oxidizing agents (e.g. K ₃ Fe (CN) ₆ )

5) mild reducing agents (e.g. dithiothreitol)

6) pH 2.4 - 9 g) it is sensitive to phenylglyoxal.

The determination of the amino acid sequence of MONAP resulted in the following structure from 72 amino acids:

SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEK-FLKRAENS

It is not yet certain _/ which part of the sequence causes the biological activity. In addition to MONAP, shorter-chain peptides can be isolated that show the same biological properties as the sequence of the 72 amino acids. Long-chain peptides of the same type can also be isolated _, such as the LYNAP with the pentapeptide terminus (AVLPR), which has been described in the meantime and which is followed by the above sequence of 72 amino acids:

(AVLPR) SAKELR ...

(Gregory, Young, Schröder, Nrowietz, Christophers, Biochem. And Biophys.

Res. Comm., Vol. 151, No. 2, March 15, 1983, pages 883-890). The analytical isoelectric focus of MONAP shows a uniform amino terminus with a charge microheterogeneity with main components with isoelectric points at 4.7, 4.9, 6.4 and 6.9, which are all chemotactically active. The determination was made according to Schröder, Mrowietz and Christophers, Biochem. and

Biophys. Res. Comm., Vol. 152, No. 1, Apr. 15, 1988, pages 277-284.

The novel biologically active polypeptide according to the invention has the following biological properties:

a) it is chemotactically and cheraokinetically active for human neutrophil granulocytes; b) it induces the release of lysosomal enzymes from cytochalasin B pretreated human neutrophil granulocytes; c) it releases super oxide anions from human neutrophil granulocytes; d) it binds to a separate receptor on human granulocytes; e) it does not activate the chemotaxis of human monocytes, eeoossiinnoopphhiilleerr GGrraannuulloozzyytteenn and LLyymphozyten (<6 x 10 ~ M) f) it has no IL-1 activity.

It is striking that the polypeptide according to the invention is stable against heating; its biological effects are not affected by heating. MONAP is in conflict here to IL-1, whose heat sensitivity is known.

The specific activity of completely purified MONAP was almost 10 half-maximum chemotaxis doses (EC ₅₀ ) / 1 mg MONAP (volume: 0.1 ml). MONAP is therefore a very effective chemotaxin for human PMNL old a calculated EC _{5 0} of nearly 5 x 10 ^{- 11} M (based on the molecular weight of 10 kD). In fact, MONAP can cause PMNL chemotaxis at doses approximately 10 to 40 times lower than C5a, which is known to have an EC _ςn of 1-3 x 10 ^-9 M.

Cross-desensitization studies using known chemotaxins (C5a, FLMP, LTB ₄ , PAF) show none

Cross-reaction with such chemotaxins.

The polypeptide according to the invention can be produced as described below.

Human mononuclear cells are stimulated, in particular with lipopolysaccharides, phythaemagglutin and / or phorbol esters, in particular phorbol myristate acetate.

The supernatant obtained is purified by gel chronography, followed by separation by HPLC cation exchange chromatography, HPLC exclusion chromatography and reversed-phase HPLC.

Monocytes which can be obtained by purifying venous blood are used in particular as mononuclear cells. Such monocytes and their purification are described, for example, by B∅yum, A. 1968, Scand. J. Clin. Lab. Invest. 21: 17. The stimulation is preferably carried out with lipopolysaccharides, phythaemagglutinin and / or phorbol esters on cell cultures of the monocytes at 37 ° C. It is in the pH range of 7-8, e.g. B. worked at 7.4. The pH value is achieved using a suitable buffer (e.g. gaseous carbon dioxide). The stimulation can e.g. B. during 24 to 40 hours. The degree of moisture is variable and depends on the cells. For example, a humidity of 95% in an air / carbon dioxide mixture (5% carbon dioxide / 95% air) can be used for 24 to 40 hours.

The supernatant obtained from the incubation is used to prepare MONAP. It is preferably first cleaned, which can be done, for example, by acidification to a pH of 2-4, in particular 4. Examples of acids are trifluoroacetic acid or formic acid. It is then centrifuged.

Gel filtration is carried out on conventional columns with Sephadex gels, at acidic to neutral pH, for example on G-75 columns (Pharmacia), for example equilibrated with ammonium formate at a pH of about 5.

HPLC cation exchange chromatography is preferably carried out in the acidified to neutral state, for example at pH 5. For example, formic acid can serve as the acid.

HPLC exclusion chromatography is preferably carried out on conventional silca columns, e.g. B. on a TSK-2000 HPLC column, at pH 2-3.

The RP-HPLC can, for example, on an organically modified silica column, e.g. B. on octadecyl silica column. The polypeptide according to the invention is chemotactically and chemokinetically active for human neutrophilic granulocytes. It is suitable for use as a pharmaceutical or for the production of pharmaceutical preparations. For systemic or local use, the formulation can be carried out in the customary manner, in the customary manner in auxiliaries and excipients.

The medicaments containing MONAP according to the invention are particularly suitable for increasing the cellular, phagocyte-associated defense against specific and non-specific inflammations through local or systemic use. In particular, MONAP has been shown to stimulate granulation tissue and wound healing when used locally or systemically. Monap can therefore, for. B. in chronic. Inflammatory processes, poorly healing wounds, fistulas, bacterial infections and chronic infections of the skin and the underlying tissues caused by mycotic pathogens are used. The application can z. B. in the form of a locally effective administration z. B. a solution.

However, MONAP can also be used as a diagnostic. The invention thus also encompasses diagnostic methods in which MONAP is used to test the reactivity of polymorphonuclear leukocytes. The test can be performed locally or systemically in vivo. However, in vitro use is also possible. For example, it can be determined whether or not leukocytes result in normal chemotaxis, enzyme release or proliferation of oxygen radicals. Another application is the use of MONAP as a reagent. For example, MONAP can be used as an antigen for the production of monoclonal antibodies. The MONAP-specific antibodies obtained and isolated in the usual way can be labeled or coupled with radioisotopes, enzymes, fluorescent or luminescent reagent it, as is known in the literature.

The antibodies labeled in this way can also be used as diagnostic agents. The invention therefore also relates to diagnostic methods using the labeled antibodies obtained using MONAP in order to determine the MONAP concentrations in tissue or blood.

The labeled antibodies obtained using MONAP can also be used for the detection of MONAP-specific receptors in tissue and blood cells. For example, it is possible to mark activated antibodies by an in vitro representation of activated leukocytes. B. to perform tissue sections or smear preparations, and to obtain information about inflammatory processes by detection in the blood.

In addition to the diagnostic utility of the MONAP-specific antibodies, the latter can also be used therapeutically. They are suitable, for example, for therapeutic methods for suppressing cellular, phagocyte-associated immune reactions, since the use of MONAP-specific antibodies or antagonists inhibits or antagonizes the effect of MONAP on the immune response of the organism.

The peptide according to the invention is therefore extremely suitable as a medicament and diagnostic agent; however, it can also be used as a reagent for the production of MONAP-specific monoclonal antibodies, which in turn can be used again for pharmaceutical and / or diagnostic purposes.

In the following a more precise specification of the practical production and characterization of MONAP is given in the form of examples.

A) Manufacture of MONAP:

1. Purification of the monocytes

The monocytes were purified according to the above-mentioned reference Bt∅yum, A. 1968. Venous blood anticoagulated with 1 aM EDTA was diluted to 1: 2 with isotonic saline solution, underlaid with Ficoll separation medium (Biochrom, Berlin) and for 35 minutes with 400 × g centrifuged at 20 ° C. The limit phase containing mononuclear cells was then obtained and the monocytes were obtained by reversible binding to human fibronectin on a gelatin-coated plastic surface according to Freundlich and Avdalovic (1983, J. Immunol. Method, 52:31).

The monocytes thus obtained showed a purity of 95 ± 4% (alpha-naphthyl acetate esterase). The contaminating cells were predominantly T-lymphocytes (2.1 ± 3%) (Leu M I monoclonal antibody staining). The monocyte preparation was Irei of neutrophils and contained less than 10% platelets. Viability over 97% (trypan blue dye exclusion test).

2. MONAP production

Peripheral blood aonocytes as obtained above were grown in tissue culture flasks (Falcon) in a humid atmosphere containing 5% CO ₂ at 37 ° C in medium 199 containing Earles salts and 20 mM HEPES (cell density = 1-2 x 10 ⁶ cells / ml). After an initial incubation of 1 hour, removal of the non-adherent cells and washing three times with medium 199, 50 ng / ml PMA were added and the monocytes were cultured for a further 2-12 hours. Then lipopolysaccharides from Salmonella minnesota RE 595 (Calbiochem, Marburg) were added (1 ng / ml) and the cultures were incubated at 37 ° C and 95% humidity in 5% CO ₂ /95% air. The conditioned mediam were collected after 24-40 hours of incubation and stored below -70 ° C until chromatographic separation.

3. Cleaning of MONAP

All cleaning operations were carried out at 4 ° C where possible. 50-700 ml of incubation supernatants were stored at -70 ° C for no longer than 2 weeks. They were thawed, adjusted to pH 4 with trifluoroacetic acid or formic acid and then by centrifugation clarified. The supernatants were concentrated 100-400 times on an Amicon YM-5 membrane and clarified by centrifugation.

4. Gel Filtration Chromatographic

3 ml of concentrated incubation medium were applied to a 2.6 × 75 cm G-75 (Pharmacia) acid equilibrated with 0.05 M ammonium formate, pH 5, it was eluted with ammonium formate at a flow rate of 10 ml / h and it 5 ml fractions were collected. Fractions with neutrophil stimulating activity (chemotaxis, enzyme release) were concentrated using Amicon YM-5 membranes and then chromatographed or frozen at -70 ° C.

The column was calibrated with the following proteins: bovine albuain 60 kD; Ovalbumin 45 kD; Myoglobulin 17 kD; Ribonuclease A 13.8 kD and cytochrome C 12.4 kD. The molecular weight of MONAP was determined on a graph of Ka _v / log (MG).

The results are shown in Table I below.

5. CM ion exchange chromatography

80-300 ml of crude supernatants acidified to pH 5 with formic acid were concentrated using Amicon YM-5 membranes and diafiltered against 0.01 M ammonium formate, pH 5.0. The samples were centrifuged, applied to a TSK CM-3SW-HPLC column (7.5 × 150 mm) previously equilibrated with 0.01 M ammonium formate and, after washing, with 10 ml of 0.01 ammonium formate, pH 5, with a 30 ml gradient eluted from 0.01 to 0.5 M ammonium formate. HPLC was at a flow rate of 1 ml / min and 1 ml fractions were collected. Samples from each fraction were tested for MONAP activity.

The results obtained are shown in Table I below.

6. DEAE ion exchange chromatography

20-80 ml of supernatants were concentrated using Amicon YM-5 membranes, diafiltered against 0.02 M Tris / sodium acetate, pH 8.0. The samples were centrifuged and applied to a TSK DEAE-5 PW-HPLC column (LKB, 7.5 × 75 mm), which had previously been equilibrated with 0.02 M Tris-acetate, pH 8 (buffer A). MONAP was eluted after washing the column with 15 ml of buffer A with a gradient (5 ml) of 0.02 M tris-acetate containing 0.5 M sodium acetate (buffer B), followed by isocratic elution (5 ml) followed and with a gradient (15 al) to 100% buffer B, followed by another isocratic elution with B.

HPLC was performed at a flow rate of 1 ml / min and 1 ml fractions were measured. Samples from each fraction were taken for testing for MONAP.

7. Exclusion HPLC (TSK 2000 HPLC)

Biologically active fractions from CM-TSK-HPLC batches or crude supernatants acidified with trifluoroacetic acid were lyophilized or concentrated to 0.3-0.5 ml and onto a TSK-2000 exclusion HPLC column (LKB, 0.8 × 60 cm), equilibrated with 0.1% (vol / vol) trifluoroacetic acid in water, applied. MONAP was eluted with 0.1% trifluoroacetic acid. The chromatography was carried out at a flow rate of 1 ml / min and fractions of 0.5 ml were collected. Samples from each fraction were taken for biological evaluation (chemotaxis or enzyme release). The column was calibrated with the following proteins (Sigma): myoglobulin 17.4 kD, ribonuclease A 13, 7 kD, cytochrome C 12, 4 kD, aprotinin 6.5 kD, insulin 3, 5 kD and tetapeptide Val Gly Ser Glu 0 , 5 kD. The molecular weights of MONAP were determined by plotting the elution time against log (MG).

8. Reversed-phase high-pressure liquid chromatography (RP-HPLC)

HPLC was performed on a chromatographic Speetra-Physics¬

System with 4, 6 × 250 mm Nucleosil - 5 μm - a Reversed¬

Phase octadecyl silica column performed. Blution was monitored using a Kratos UV detector at 215 ml. The peak areas were determined by a Spectra-Physics

SP 4270 integrator calculated.

Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in acetonitrile. The column was equilibrated with 10% B in A. Fractions from TSK 2000-HPLC containing MONAP were pooled, concentrated and injected onto the column. MONAP was eluted with a 2-slope gradient: 10 to 40% B in 30 minutes, followed by 40 to 100% B in 10 minutes. Peak fractions were collected manually. Samples were taken for SDS-PAGE as well as for MONAP and LAF / IL-1 bioassay. The samples were stored below -70 ° C or lyophilized and redissolved in suitable buffers for further use. 9. Polyacrylamide gel electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for peptides was performed as described by Swank and Munkres, 1971, Anal. Biochem. 39: 462, performed using 13-lane, 160 x 120 x 1.50 mm gels, with 13.8% T, 9.1% C, in the presence of 8 M urea. 0.1M phosphoric acid, adjusted to pH 6.8 with Tris, containing 1% (w / v) SDS, was used as the electrophoresis buffer. The proteins were visualized by silver staining (Amersham, Braunschweig). The following proteins were used as the molecular weight standard: polypeptide molecular weight calibration kit (Pharmacia, Freiburg), containing sperm whale myoglobulin 17.2 kD; partial cyanogen bromide cleavage products I + II 14.6 kD; I 8.2 kD; II 6.4 kD; III 2.5 kD; Myoglobulin 1-14 1.7 kD, trypsinogen, PMSF treated, 24 kD; α-lactalbumin 14.2 kD; recombinant human tumor necrosis factor (TNF) 17 kD and cytochrome C 12.4 kD. The molecular weight of MONAP was determined from an application of R _f against log (MG).

10. Results

Production of the PBM supernatants

Processing 500 ml of blood resulted in purified monocytes for the preparation of 100 ml of MONAP-enriched supernatants, which typically contained 8-12 × 10 ⁴ doses to stimulate the half-maximum PMNL chemotaxis (EC ₅₀ ). The protein content, activity and calculated yield for each stage of MONAP purification are given in Table I below.

Purification of human MONAP

Studies have shown that the concentration of raw supernatants are best carried out at pH 3-4.

Passing acidified supernatants through Sephadex G-75 using 0.05 M ammonium formate, pH 5, as the elution buffer was more effective than using PBS. In cation exchange chromatography, the MONAP activity was eluted in fractions 11-18. Chromatography removed 90% of the protein.

In HPLC exclusion chromatography using the TSK 2000 SW column, MONAP activity was eluted in a single peak with neutrophil stimulating activity.

RP-HPLC showed a single peak. SDS-PAGE of this material showed a single line corresponding to a molecular weight of 10 kD. This molecular weight was also determined by TSK 2000 HPLC.

B) Characterization of MONAP:

MONAP assays:

Neutrophil stimulating activity (PMNL) was measured in three ways:

Chemotactic PMNL activity of samples was measured using the method of Creamer et al., 1983, Inflammation 7: 321. Boyden chambers (Bio Rad, Munich) were filled with suitably diluted samples, covered with a polyvinyl pyrrolidone-free polycarbonate filter (pore size 3 μm) and with human PMNL, suspended in PBS, with a content of 0.9 mM CaCl ₂ , 0.5 mM MgCl ₂ and 1% BSA filled.

The Boyden chambers were incubated in a humid atmosphere at 37 ° C for 1 hour. The cover and filter were then carefully removed and migrated Cells remaining in the lower part of the Boyden chamber were dissolved by adding 10 µl of 1% Triton X-100 (vol / vol) and then incubating for 10 minutes. The entire content (110 μl) was incubated with 100 μl 0.01 M p-nitrophenyl-β-glucuronide (Sigma, Munich) in 0.1 M sodium acetate buffer, pH 4.0, 18 hours and the enzymatic reaction was carried out by suspension of 200 ul 0.6 M glycine buffer, pH 10, stopped. p-Nitrophenolate was determined at 405 nm using a multi-channel photoneter (SLT 210, Kontron). For the calibration, dissolved PMNL (5 × 10 ^-3 - 2 × 10 ⁴ cells) in 110 μl PBS with a content of 0.1% (vol / vol) Triton X-100) were incubated with the β-glucuronidase substrate.

Chemotactic activity was expressed in PMNL equivalents, which migrated through the filter in 1 hour. Defined chemotaxins (C5a, 50 ng / ml, FMLP 4 × 10 ^-9 M, LTB ₄ 1 ng / ml) were used as controls.

The checkerboard analysis of chemokinetic PMNL activity was performed by adding defined concentrations of MONAP to the top and bottom of the Boyden chamber. The number of cells migrated was shown as a function of the MONAP concentration in the upper and lower chambers.

2. Chemotaxis examinations

Meutrophilic chemotaxis and cheaokinesis studies were performed as described above.

The eosinophilic chemotaxis was measured in the same way using a nitrocellulose filter with a 0.45 μm pore size, followed by a polycarbonate filter (upper filter, nucleopore, pore size 3 μm) placed at the top End of the lower part of a Boyden chamber. Eosinophilic suspensions containing 10 cells / ml PBS / BSA were placed in the upper part of the chamber.

The Boyden chamber was then incubated for 2 hours at 37 ° C in a humid atmosphere. The cells adhering to the nitrocellulose filters were fixed with methanol and according to the method of Kay, 1970, Clin. exp. Immunol. 7: 723, stained using hematoxylin and Chromotrop 2R and attached to glass slides. Cells from 5 statistically evaluated high power fields were counted under a microscope at 400x magnification and the average of two examinations was shown as cells per visual field.

Monocyte chemotaxis and statistical migration were examined using the Boyden chamber system using a double membrane filter technique with indirect quantitative evaluation of the migrated cells. Cytoplasmic lactate dehydrogenase was used as the labeling enzyme.

The chemotactic activity was presented as a chemotactic index (CI).

3. Enzyme release

PMNL (10 ⁷ / ml PBS) was preincubated with cytochalasin B (5 μg / ml, Sigma). Then 100 μl samples with suitable dilutions were added and incubated for a further 30 minutes. After centrifugation, 100 μl was incubated for 18 hours with 100 μl of 0.01 M p-nitrophenyl-β-D-glucuronide in 0.1 M sodium acetate, pH 4. The enzyme reaction was stopped by adding 200 μl 0.4 mol / l glycine buffer, pH 10. The β-glucuronidase release was shown as a percentage of an overall control, with 0.2% (vol / vol) Triton X-100 being added instead of a stimulus (= 100% control).

In some cases, rapid determination of PMNL enzyme release activity was performed by testing stimulated cell supernatants for myeloperoxidase activity (MPO). 100 ul supernatants were incubated with 100 ul 0.01 M o-phenylenediamine dihydrochloride in 0.1 M citrate / phosphate buffer, pH 5, containing 0.001% hydrogen peroxide for 10 minutes at room temperature. The enzyme reaction was stopped by adding 100 μl of 2 M sulfuric acid and the brownish color was determined in microtiter plates at 485 nm using a multi-channel photometer. Suitable

Controls were obtained by incubating cells with C5a (100 ng / ml), FMLP (4 × 10 ^-7 M), 0.1% hexadecyltrimethylammonium bromide (= 100% control). The lactic acid dehydrogenase release was determined as a control of cell integrity, which in no case exceeded 3% of the total control.

Enzyme release activity was expressed either as OD ₄₈₆ or as a percentage of the 100% control.

4. Desenibilization examinations

The cross-activities of PMNL cheaotaxin receptors were examined by preincubating 2 × 10 ⁷ PMNL alt different chemotaxins at optimal concentrations (C5a: 100 ng / ml; PAF 3 × 10 ^-6 M; BOC-Met-Leu-Phe 10 ^{- 5} M; LTB ₄ 10 ^-8 M; MONAP ^{10-1 0} M buffer) for 20 minutes at 37 ° C. This was followed by the addition of cytochalasin B (5 µg / ml) followed by incubation for 5 minutes at 37 ° C. The cells were then stimulated with various stimuli (C5a, 100 ng / ml; FMLP 4 × 10 ^-8 M; PAF 10 ^-6 M; MONAP 10-1 ⁰ M; LTB ₄ 10 ^-8 M, or buffer) and others Incubated for 30 minutes. Finally, the β-glucuronidase was determined.

5. Generation of Superoride Anions (O- ₂ )

250 μl PMNL suspension (8 × 10 ⁶ / ml PBS) containing 0.8 mM CaCl ₂ , 0.5 mM MgCl ₂ and 0.1% BSA) were mixed with 5 μg / ml cytochalasin B (5 minutes at 37 ° C) and then treated with 500 µl of oxidized cytochrome C (Sigma) in PBS / 0.1% BSA and 250 µl of the appropriate dilution of the stimulus, each preheated to 37 ° C. After 20 minutes, O- ₂ generation was stopped by cooling in ice / water, followed by centrifugation (400 g / 10 min). The reduced cytochrome C in the supernatants was assessed by measuring the absorbances at 474.4 nm and 549.1 nm using vs. Blank samples containing 10 μg superoxide dismutase at the start of the study. The O- ₂ concentrations were calculated by evaluating the SOD-inhibitable cytochrome C reduction after calibration of the

Spectro-photometers with completely reduced (sodium dithionite) and completely oxidized cytochrome C.

6. Results

The effect of various agents and influences on MONAP activity is shown in Table II. MONAP was stable at pH 2.4 - 9 and under mild oxidizing and reducing conditions. In addition, PMSF showed no change in biological activity. However phenylglyoxal and an arginine-reactive reagent reduced biological activity.

The biological activity of MONAP was not, or hardly changed, by heating to 60 ° C and 100 ° C.

Purified MONAP could be frozen, stored at 4 ° in 0.1% trifluoroacetic acid, pH 2.4, or lyophilized only with a slight loss of activity. However, storage without additional protein in PBS at 4 ° C (18 hours) resulted in a drastic loss of activity, evidently by binding to the plastic material.

The dose-response curves of purified MONAP in the PMNL chemotaxis assay, in the enzyme (β-glucuronidase) release assay and in the superoxide anion release assay showed half-maximum stimulation of the PMNL cheaotaxis at 5 × 10 ^{-1 1} M MONAP. MONAP's checkerboard analysis demonstrated chemokinetic activity in addition to chemotactic activity (Table III). The half-maximal release of azurophilic granule enzymes after pretreatment of PMNL with cytochalasin B took place at 2 x 10 ^-10 M.

O- ₂ generation, represented by cytochrome C examination, is activated in a dose-dependent manner by MONAP. However, the amounts of O ₂ - released are relatively small and could only be determined using high amounts of PMNL, pretreated with cytochalasin B.

In no case did highly purified MONAP preparations show IL-1 activity; in addition, IL-1, isolated in partially purified form from the RP-18 column, showed no neutrophil stimulating activity. To determine whether MONAP binds to chemotaxin receptors of known chemotaxins, cross-desensitization of PMNL reactions after preincubation of PMNL with chemotaxins was carried out. The results showed that the MONAP reactions of PMNL were reduced only when the lines were preincubated with MONAP, but not with the other chemotaxins mentioned.

It was also found that neither human monocytes nor eosinophils could be stimulated to chemotactically migrate in concentrations up to 6 x 10 ^-10 M, demonstrating the specific neutrophil activity of MONAP under these conditions.

The neutrophil activating peptide can be applied in a locally effective administration, e.g. B. as a solution for chronic inflammatory processes, poorly healing wounds, fistulas, bacterial infections and chronic infections caused by mycotic pathogens, the skin and the underlying tissues. The strong leukotactic effect leads to cleaning and subsequent healing of the areas.

Another area of application extends to the production of monoclonal antibodies for diagnostic purposes. It is possible, by labeling the antibodies, for an in vitro display of activated leukocytes, for. B. to perform tissue sections or smear preparations, and to obtain information about inflammatory processes by detection in the blood. C) Dustibility studies:

To investigate the chemical stability, 10 μl of test agents, which are listed below, were added to 100 μl of highly purified MONAP in PBS: 10 mM dithiothreitol, 10 mM sodium dithionite, 1 mM potassium ferricyanide, 10 mM phenylglyoxal and 10 mM phenylmethylsulfonyl fluoride. After 1 hour at 37 ° C, the samples, including a buffer control sample, were diafiltered with 3 ml PBS containing 0.1% BSA on an Amicon YM-5 membrane and half the maximum dose causing neutrophil chemotaxis ( EC ₅₀ ) was evaluated by determining five dilutions (in duplicate).

The results obtained are shown in Table II.

Claims

1 . Neutrophil activating polypeptide

isolable from human mononuclear cells stimulated with lipopolysaccharides, phythaemagglutin and / or phorbol esters;

- With a molecular weight of 10,000 Dal ton

- detectable by a single band in sodium dodecyl sulfate-polyamide gel electrophoresis and

- detectable by a single peak at the

Exclusion high pressure liquid chromatography; and

- with a single peak in reversed-phase high pressure liquid chromatography.

2. The method for producing the neutrophil-activating polypeptide according to claim 1, characterized by the following steps: a) stimulation of human mononuclear cells with lipopolysaccharides, phythaemagglutin and / or phorbol esters, b) separation of the supernatant by means of gel chromatography, e) further separation by cation exchange high-pressure liquid chromatography acidic conditions, d) subsequent exclusion high pressure liquid chromatography and e) reversed phase high pressure liquid chromatography

3. Drugs containing the neutrophil activating

Polypeptide according to claim 1, in addition to conventional pharmaceutically acceptable auxiliaries and carriers.

4. Use of the neutrophil activating polypeptide according to claim 1 as a diagnostic.

5. Use of the neutrophil activating polypeptide according to claim 1 for the production of monoclonal antibodies.