DE3737703A1 - NEUTROPHILE ACTIVATING POLYPEPTIDE, METHOD FOR THE PRODUCTION THEREOF AND THE USE THEREOF AS A MEDICINAL PRODUCT AND DIAGNOSTIC - Google Patents

Abstract

A neutrophil-activating polypeptide isolated from stimulated human mononuclear cells has a molecular weight of 10 000 daltons and a single peak in reversed-phase high-performance liquid chromatography. In the process for manufacturing the polypeptide, human mononuclear cells are stimulated with lipopolysaccharides, phythaemaglutin and/or phorbol esters and the excess is separated by gel chromatography. Further separation is carried out by cation-exchange high-performance liquid chromatography, exclusion high-performance liquid chormatography, and reversed-phase high-performance liquid chromatography. The polypeptide can be used locally or systemically to increase the cellular phagocyte-associated defence and is therefore useful as a drug. It can also be used as a diagnosticum and as a reagent for the production of monoclonal antibodies.

Description

The invention relates to a neutrophil activating polypeptide, isolated from human mononuclear cells. This polypeptide is active for human neutrophil granulocytes and is therefore suitable as a pharmaceutical, in particular to increase the cellular phagocyte-associated defense inflammation and stimulation of granulation tissue and wound healing. It is also used as a diagnostic for testing the reactivity of neutrophils polymorphonuclear leukocytes in vivo or in vitro as well as Reagent for producing monoclonal antibodies using suitable as an antigen.

A polymorphonuclear nucleus released by human monocytes Leukocyte (PMNL) activating factor is known. This Factor appears in addition to chemotaxis (T. Tono-oka et al., 1980, Immunology 39: 607) PMNL degranulation (Klemper et al., 1978, J. Clin. Invest. 61: 1330) and the oxygen-dependent Activate metabolism (Klempner et al., 1979, J. Clin. Invest. 64: 996). Subsequent investigations showed that partial purified interleukin-1 preparations stimulate this PMNL Show 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 was a PMLN activating cytokine represents.

IL-1 denotes a series of biochemically closely related cytokines, which are generated by different cell types and includes the Lymphocyte activating factor (LAF), endogenous pyrogen (EP), endogenous leukocyte mediator (LEM) or the mononuclear  Cell factor (MCF). It is believed that these factors numerous biological effects such as fever cause Proliferation of fibroblasts, the production of acute Phase proteins that stimulate the catabolism of Muscle protein and the neutrophil chemotaxis (Dinarello, C.A. 1896, The Year in Immunology 2: 68).

IL-1 activity has been demonstrated in various biochemical Forms described, d. H. 17 kD species with IP 6.8, 5.4 and 5.2, and high molecular weight forms with similar ones IP values. However, it is still not clear whether the biological properties of IL-1 of a single molecular species may or may not be attributed to IL-1.

Within the scope of the present invention, a neutrophil has now been developed activating peptide (hereinafter also called MONAP identified) found that differs from IL-1.

The invention therefore relates to a new, physiologically active Polypeptide with a selective chemotactic effect neutrophilic, polymorphonuclear leukocytes (MONAP). It relates also refer to the process for its extraction, as well medicinal product containing it and its use.

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

The polypeptide according to the invention can be chemico-physically define as follows:  

• a) it is a polypeptide;
• b) Molecular weight determined by SDS electrophoresis: 10 kD;
• c) determined by exclusion chromatography (TSK-2000-HPLC) Molecular weight, also 10 kD;
• d) bind to anion exchanger 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. K₃ Fe (CN) ₆)
  • 5) mild reducing agents (e.g. dithiothreitol)
  • 6) pH 2.4-9
• g) it is sensitive to phenylglyoxal.

The novel biologically active polypeptide has following biological properties:

• a) it is chemotactically and chemokinetically active for humans neutrophil granulocytes;
• b) it induces the release of lysosomal enzymes Cytochalasin B pretreated human neutrophils Granulocytes;
• c) it sets superoxide anions from human neutrophils Granulocytes free;
• d) it binds to a separate receptor on human Granulocytes;
• e) it does not activate the chemotaxis of human monocytes, eosinophilic granulocytes and lymphocytes (<6 × 10 ^-10 M);
• f) it has no IL-1 activity.

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

The specific activity of completely purified MONAP was almost 10⁶ half-maximal chemotaxis doses (EC₅₀) / 1 mg MONAP (volume: 0.1 ml). MONAP is therefore a very effective chemotaxin for human PMNL with a calculated EC₅₀ of almost 5 × 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 EC₅₀ of 1-3 × 10 ^-9 M.

Cross-sensitization studies using known chemotaxins (C5a, FLMP, LTB₄, PAF) show none Cross-reaction with such chemotaxins.

The polypeptide of the invention can be as follows described.

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

The supernatant obtained is purified by gel chromatography, whereupon separation by HPLC cation Exchange chromatography, HPLC exclusion chromatography and Connect reversed phase HPLC.

Monocytes in particular are used as mononuclear cells, obtained by purifying venous blood can. Such monocytes and their purification for example described by Bøyum, A. 1968, Scand. J. Clin. Lab. Invest. 21: 17.  

The stimulation is preferably carried out with lipopolysaccharides, Phythaemagglutin in and / or phorbol esters on cell cultures of the Monocytes at 37 ° C, a humidity of 95% in one Air / carbon dioxide mixture (5% carbon dioxide / 95% air) during 24 to 40 hours.

The supernatant obtained from the incubation is used for production used by MONAP. It is preferably cleaned first, for example by acidifying with trifluoroacetic acid or formic acid to pH 4 and subsequent centrifugation can be done.

Gel filtration takes place, for example, on G-75 columns (Pharmacia), for example equilibrated with ammonium formate at a pH of around 5.

HPLC cation exchange chromatography is preferred in the acidified state, for example at pH 5. As acid can serve, for example, formic acid.

HPLC exclusion chromatography is preferably carried out on one TSK-2000 HPLC column at pH 2-3.

RP-HPLC can be used, for example, on an octadecyl silica Column.

The polypeptide according to the invention is chemotactic and chemokinetic active for human neutrophil granulocytes. It is suitable for use as a pharmaceutical or for manufacturing pharmaceutical preparations. The wording can be systemic or local application in the usual way with usual Auxiliaries and carriers are used.

The medicaments containing MONAP according to the invention are particularly suitable for increasing the cellular, phagocyte associated defense against specific and nonspecific Inflammation from local or systemic use.  In particular, it has been shown that MONAP is the granulation tissue and wound healing in local or systemic Application stimulates. MONAP can therefore e.g. B. in chronic Inflammatory processes, poorly healing wounds, fistulas, bacterial infections and chronic through mycotic Pathogen-related infections of the skin and the underlying Tissues are used. The application can e.g. B. in the form of a locally effective administration z. Legs Solution.

However, MONAP can also be used as a diagnostic. The invention thus also makes diagnostic methods includes where MONAP is used to increase responsiveness to test polymorphonuclear leukocytes. The The test can be performed locally or systemically in vivo. However, in vitro use is also possible.

Another application is in use from MONAP as reagent. So MONAP can be used as an antigen for production monoclonal antibodies can be used. In the MONAP-specific obtained and isolated in the usual way Antibodies can with radio as known in the literature isotopes, enzymes, fluorescent or luminescent Reagents are marked or coupled.

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

The labeled antibodies obtained using MONAP can also be used for the detection of MONAP-specific Use receptors in tissue and blood cells. For example it is possible to label an antibody in vitro representation of activated leukocytes z. B. on Perform tissue cuts or cut-out specimens, as well as evidence of inflammation in the blood to obtain.  

Apart from the diagnostic usability of the MONAP specific antibodies can also be used therapeutically be used. For example, they are suitable for therapeutic methods for the suppression of cellular, Phagocyte-associated immune reactions, because of the Use of MONAP-specific antibodies or antagonists the effect of MONAP on the immune response of the organism is inhibited or antagonized.

The peptide according to the invention is therefore extremely suitable as a medicine and diagnostic agent; however, it can also be used as Reagent for the production of MONAP-specific monoclonal Antibodies are used, which in turn are used for pharmaceutical and / or diagnostic use can find.

The following is a more detailed specification of the practical Manufacture and characterization of MONAP in form given by examples.

A) Manufacture of MONAP 1. Purification of monocytes

The monocytes were purified according to the above Bøyum, A. 1968. With 1 mM EDTA anticoagulated venous blood was mixed with isotonic saline diluted to 1: 2, with Ficoll separation medium (biochrom, Berlin) and centrifuged at 400 × g at 20 ° C for 35 minutes. Then the mononuclear cell containing Boundary phase won and the monocytes were through reversible binding to human fibronectin on a Gelatin-coated plastic surface according to Freundlich  and Avdalovic (1983, J. Immunol. Methods, 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 MI monoclonal antibody staining). The monocyte preparation was neutrophil free and contained less than 10% platelets. Viability over 97% (trypan blue dye Exclusion test).

2. MONAP production

Peripheral blood monocytes as obtained above were found in Tissue cultures (falcon) in a humid atmosphere a content of 5% CO₂ at 37 ° C in medium 199 with a Content of Earles salts and 20 mM HEPES (cell density = 1-2 × 10⁶ cells / ml) introduced. After initial incubation of 1 hour, removing the non-adherent cells and triple Washes with medium 199, 50 ng / ml PMA were added and the monocytes were cultured for an additional 2-12 hours. Subsequently, lipopolysaccharides from Salmonella minnesota RE 595 (Calbiochem, Marburg) added (1 µg / ml) and cultures were grown at 37 ° C and 95% humidity in 5% CO₂ / 95% air incubated. The conditioned media were collected after 24-40 hours of incubation and below -70 ° C stored until chromatographic separation.

3. Cleaning MONAP

All cleaning processes were, as far as possible, at 4 ° C carried out. 50-700 ml of incubation supernatants were added no longer than 2 weeks at -70 ° C. they were thawed with trifluoroacetic acid or formic acid pH 4 adjusted and then by centrifugation  clarified. The supernatants were increased to 100-400 times an Amicon YM-5 membrane and concentrated Centrifugation clarified.

4. Gel filtration chromatography

3 ml of concentrated incubation medium was applied to a 2.6 × 75 cm G-75 (Pharmacia) acid equilibrated with 0.05 M ammonium formate, pH 5, applied. It was made with ammonium formate eluted 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 albumin 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 in Table I below listed.

5. CM ion exchange chromatography

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

The results obtained are in Table I below listed.

6. DEAE ion exchange chromatography

20-80 ml of supernatants were concentrated using Amicon YM-5 membranes, against 0.02 M Tris / sodium acetate, pH 8.0, diafiltered. The samples were centrifuged and on a TSK DEAE-5 PW-HPLC column (LKB, 7.5 × 75 mm), the previously equilibrated with 0.02 M Tris-acetate, pH 8 (buffer A) had been applied. MONAP became 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) eluted, followed by an isocratic elution (5 ml) followed and with a gradient (15 ml) 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 collected. Samples from each fraction were tested for MONAP taken.

7. Exclusion HPLC (TSK 2000 HPLC)

Biologically active fractions from CM-TSK-HPLC batches or crude supernatants acidified with trifluoroacetic acid lyophilized or concentrated to 0.3-0.5 ml and on a TSK-2000 exclusion HPLC column (LKB, 0.8 × 60 cm), equilibrated with 0.1% (vol / vol) trifluoroacetic acid in Water, applied. MONAP was made with 0.1% trifluoroacetic acid eluted.  

Chromatography was at a flow rate of 1 ml / min and fractions of 0.5 ml collected. Samples from each fraction were biological Assessment (chemotaxis or enzyme release) taken. The 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 tetrapeptide Val Gly Ser Glu 0.5 kD. The molecular weights of MONAP were from an order of the elution time against log (MG) determined.

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

HPLC was performed on a Spectra-Physics chromatographic System with 4.6 × 250 mm Mucleosil - 5 µm - a reversed Phase octadecyl silica column performed. The elution was measured using a Kratos UV detector at 215 nm supervised. 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 containing TSAP 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 made for SDS-PAGE as well as for MONAP and LAF / IL-1 bioassay. The samples were stored below -70 ° C or lyophilized and again for further use in suitable buffers solved.  

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. 0.1 M phosphoric acid, adjusted to pH 6.8 with Tris, with a content of 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 production of 100 ml enriched with MONAP Supernatants, typically 8-12 × 10⁴ doses to stimulate the semi-maximum PMNL chemotaxis (EC₅₀) contained. The protein content, the activity and the calculated Yield for each stage of MONAP cleaning are in given in Table I below.

Purification of human MONAP

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

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

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

RP-HPLC showed a single peak. SDS-PAGE this Materials gave a single line corresponding to one 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 CaCl₂, 0, 5 mM MgCl₂ 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 of 0.01 M p-nitrophenyl- β- glucuronide (Sigma, Munich) in 0.1 M sodium acetate buffer, pH 4.0, for 18 hours and the enzymatic reaction was carried out by adding 200 µl 0.4 M glycine buffer, pH 10, stopped. p-Nitrophenolate was determined at 405 nm using a multi-channel photometer (SLT 210, Kontron). For the calibration, dissolved PMNL (5 × 10 ^-3 - 2 × 10⁴ 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, LTB₄ 1 ng / ml) were used as controls.

Checkerboard analysis determination of chemokinetic PMNL activity was carried out by adding defined Concentrations of MONAP to the top and bottom Part of the Boyden Chamber. The number of cells migrated was determined as a function of the MONAP concentration in the shown upper and lower chamber.

2. Chemotaxis examinations

Neutrophil chemotaxis and chemokinesis studies were carried out as described above.

The eosinophilic chemotaxis was measured in the same way using a 0.45 µm nitrocellulose filter 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. Eosinophils Suspensions containing 10⁶ cells / ml PBS / BSA were placed in the upper part of the chamber.

The Boyden chamber was then run at 37 ° C for 2 hours incubated in a humid atmosphere. The on the nitrocellulose filter adhering cells were fixed with methanol and by the method of Kay, 1970, Clin. exp. Immunol. 7: 723, using hematoxylin and Chromotrop 2R colored and fastened on glass supports. Cells of 5 statistically High power fields were rated under one Microscope counted at 400x magnification and the mean of two studies was done as cells per visual field shown.

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

The chemotactic activity was called the chemotactic index (CI).

3. Enzyme release

PMNL (10⁷ / PBS) was treated with cytochalasin B (5 µg / ml, Sigma) pre-incubated. Then 100 ul samples with suitable Dilutions were added and incubated for a further 30 minutes.  

After centrifugation, 100 μl of supernatant were 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 ul 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 myeloperoxidase activity (MPO) activity by stimulated cell supernatants. 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% hexadecyltrimethyl ammonium bromide (= 100% control). Lactic acid dehydrogenase release was determined as a cell integrity control that never exceeded 3% of the total control.

The enzyme release activity was either as OD₄₈₆ or expressed as a percentage of 100% control.

4. Desensitization studies

The cross-activities of PMNL chemotaxin receptors were examined by preincubating 2 × 10 ^-7 PMNL with different chemotaxins at optimal concentrations (C5a: 100 ng / ml; PAF 3 × 10 ^-6 M; BOC-Met-Leu-Phe 10 ^-5 M; LTB₄ 10 ^-8 M; MONAP 10 ^-10 M buffer) for 20 minutes at 37 ° C. This was followed by the addition of cytochalasin B (4 µg / ml) followed by incubation for 5 minutes at 37 ° C. Then the cells were stimulated with various stimuli (C5a, 100 ng / ml; FMLP 4 × 10 ^-8 M; PAF 10 ^-6 M; MONAP 10 ^-10 M; LTB₄ 10 ^-8 M, or buffer) and another 30 minutes incubated. Finally the β- glucuronidase was determined.

5. Generation of superoxide anions (O ^- ₂)

250 μl PMNL suspension (8 × 10⁶ / ml PBS) containing 0.8 mM CaCl₂, 0.5 mM MgCl₂ and 0.1% BSA) were mixed with 5 µg / ml cytochalasin B (5 minutes at 37 ° C ) pretreated and then mixed with 500 µl oxidized cytochrome C (Sigma) in PBS / 0.1% BSA and 250 µl of the appropriate dilution of the stimulus, preheated to 37 ° C. After 20 minutes, the 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 spectrophotometer with completely reduced (sodium dithionite) and completely oxidized cytochrome C.

6. Results

The effect of various means and influences on the MONAP activity is shown in Table II. MONAP was stable at pH 2.4-9 and under mild oxidizing and reducing conditions. Furthermore revealed PMSF did not change biological activity. However  decreased phenylglyoxal and an arginine-reactive reagent the biological activity.

By heating to 60 ° C and 100 ° C the biological MONAP activity not or almost unchanged.

Cleaned MONAP could be frozen, at 4 ° in 0.1% Trifluoroacetic acid, pH 2.4, can be stored or only under low activity loss can be lyophilized. A storage without additional protein in PBS at 4 ° C (18 hours) resulted in a drastic loss of activity, obviously 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 chemotaxis at 5 × 10 ^-11 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 × 10 ^-10 M.

The O ^- ₂ generation, represented by cytochrome C examination is activated by MONAP in a dose-dependent manner. 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 one IL-1 activity; also showed IL-1, isolated in part purified form, from the RP-18 column, no neutrophils stimulating activity.  

To determine if MONAP is known at chemotaxin receptors Chemotaxine binds have been cross-desensitizations of PMNL reactions after preincubation of PMNL with chemotaxins carried out. The results showed that the MONAP Responses from PMNL were reduced only when the Cells 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 at concentrations up to 6 x 10 ^-10 M, demonstrating the specific neutrophil activity of MONAP under these conditions.

The neutrophil activating peptide can be used in a locally effective administration e.g. B. as a solution chronic inflammatory processes, poorly healing wounds, Fistulas, bacterial infections and chronic through mycotic pathogen-related infections of the skin and the underlying tissue. Due to the strong leukotactic Effect comes after cleaning and subsequent Healing of the areas.

Another area of application extends to Production of monoclonal antibodies for diagnostic purposes. It is possible to mark a by labeling the antibodies in vitro representation of activated leukocytes z. B. on Perform tissue cuts or smear preparations, as well by detecting in the blood information about inflammatory processes receive.  

C) Stability studies

10 µl were used to investigate the chemical stability Test agents listed below, 100 µl highly purified MONAP added in PBS: 10 mM dithiothreitol, 10mM sodium dithionite, 1mM potassium ferricyanide, 10mM Phenylglyoxal and 10 mM phenylmethylsulfonyl fluoride. To Samples, including one, were run at 37 ° C for 1 hour Buffer control sample, with 3 ml PBS containing 0.1% BSA on an Amicon YM-5 membrane and diafiltered half the maximum dose that a neutrophil chemotaxis caused (EC₅₀), was determined by determining five dilutions (in double) rated.

The results obtained are shown in Table II.  

Table I

Purification of human MONAP

Table II

MONAP stability

Table III

Checkerboard analysis of the neutrophil migration activity stimulated by MONAP ^a) upper chamber ^b)

Claims (5)

1. Neutrophil activating polypeptide

• isolated from human mononuclear cells stimulated with lipopolysaccharides, phythaemagglutin and / or phorbol esters;
• - With a molecular weight of 10,000 daltons
  • - as evidenced by a single band in sodium dodecyl sulfate polyamide gel electrophoresis and
  • - as evidenced by a single peak in exclusion high pressure liquid chromatography;
  • and
• - With a single peak in reversed-phase high pressure liquid chromatography.

2. A method for producing the neutrophil activating polypeptide according to claim 1, characterized by the following steps:

• a) stimulation of human mononuclear cells with Lipopoly saccharides, phythaemagglutin and / or phorbol esters,
• b) separation of the supernatant by means of gel chromatography,
• c) further separation by cation exchange high pressure liquid chromatography under 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, besides usual pharmaceutical compatible auxiliaries and carriers.   4. Use of the neutrophil activating polypeptide after Claim 1 as a diagnostic. 5. Use of the neutrophil activating polypeptide after Claim 1 for the production of monoclonal antibodies.