CHEMOTACTIC FACTOR
The present invention relates to a new chemotactic factor and to the use of said factor for application in tissue repair, infection and neoplasia. Background of the invention Cells of the monocyte-macrophage lineage (also called mononuclear phagocytes) play a central role in inflammation, immunity and tissue repair (1). Mononuclear phagocytes derive from precursors in the base marrow whose proliferation and differentiation is under the control of a family of glycoproteins known as colony stimulating factors (2). At the stage of monocytes, mononuclear phagocytes are released in the circulation.
Monocytes circulate in blood with a half life of 8-20h and from the blood these cells extravasate to tissues, where their life span has not been accurately determined but is of the order of months. The regulation of monocyte extravasation is a crucial determinant of inflammatory and immunological processes. Various factors are involved in regulating the recruitment in tissues of cells of the monocyte- macrophage lineage (3, 4). These include the regulated expression of adhesion molecules on vascular endotheliu that bind circulating monocytes and the production in the vessel wall or in the tissue of molecules which induce directional migration (chemotaxis) of monocytes.
Once in tissues mononuclear phagocytes can exert pleiotropic functions. By phagocytosing particles
and/or releasing toxic products mononuclear phagocytes can dispose of microbial organisms. In addition, monocytes can release molecules which partecipate in tissue destruction and remodelling, including proteolytic enzymes and enzyme inhibitors.
The extracellular matrix is in a dynamic equilibrium between synthesis of its components and degradation. Mononuclear phagocytes, central cells in the inflammatory process, are potent producers of proteinases, including collagenase and gelatinase (5- 7), which are also released by connective tissue elements. These lytic enzymes, of different cellular sources, are important for invasion by tumors and for the tissue destruction which represents an essential component of degenerative and inflammatory diseases.
In addition to partecipating to tissue destruction in pathology, mononuclear phagocytes, by secreting a variety of growth factors such as platelet-derived growth factor (PDGF) and the fibroblast growth factors (FGF) (8) in copious amounts, can also drive tissue reconstitution and repair.
In the context of strategies aimed at influencing the reconstitution and repair of damaged tissues it may therefore be desirable to identify a molecule which attracts monocytes and concomitantly inhibits the activity of tissue destructive enzymes. Summary of the invention
We have purified to homogeneity and obtained a partial aminoacid sequence of a polypeptide which induces directional migration of monocytes.
The partial sequence of 17 aminoacids is identical
to that of tissue inhibitor of metalloproteinase 1 (9). The molecule inhibits gelatinase and, concomitantly, induces chemotactic migration of monocytes. This dual function of this molecule indicates a novel utilization for the promotion of tissue repair. Brief description of the Figures Figure 1
SDS-PAGE analysis of HPLC column. Fractions were run (20 μl/lane) under reducing conditions and stained with silver. The peak of the monocyte chemotactic activity resulted in fractions 70 and 71. SDS-PAGE analysis of these fractions reported the presence of a pure polypeptide with an apparent molecular weight of 25 kDa. Figure 2
NH2 - terminal sequence analysis of fractions 70 and
71.
These fractions containing a pure polypeptide were subjected to sequence analysis by an Applied Biosystems 477A/120A sequencing system. It was optained a single sequence that fully corresponds with human tissue inhibitor of metalloproteinases-1 (hTIMP-1) (9). Description of the invention
According to the present invention, we have purified and molecularly characterized a polypeptide released by an ovarian cancer cell line and able to induce directional migration of monocytes. The supernatant of the SW 626 ovarian cell line was prepared as described in Example 1. It was found to contain a monocyte specific chemoattractant, as determined by the method described in Example 2. The
supernatant was subjected to a purification protocol, described in Example 3.
This protocol of separation resulted in a pur polypeptide with an apparent molecular weight of 25 kDa on SDS-PAGE (Figure 1).
The fractions containing the pure polypeptide were subjected to sequence analysis by an Applied Biosystems 477A/120A sequencing system.
The sequence is shown in Figure 2. The sequence of the SW 626 chemoattractant is identical to that of TIMP-1 from aminoacid 4 to aminoacid 12 and from aminoacid 13 to aminoacid 20.
The capacity of the SW 626 chemoattractant to inhibit metalloproteinases (a typical activity of TIMP- 1) was verified by reverse casein zymography using MMP-
3 from MDA 231 breast carcinoma cell line as the metalloproteinase to be inhibited.
As shown in Table 1 the SW 626 chemotactic factor (CF/TIMP) , induced migration of monocytes but not of neutrophils. This kind of activity has never been reported for the known TIMP.
The specific activity of CF/TIMP of the invention was calculated defining units as the amount of material required to induce half maximal migration of monocytes. CF/TIMP had a specific activity of approximately 6000 μ/mg protein.
CF/TIMP of the present invention, in view of its novel activity described herein consisting of the capacity to induce directional migration of monocytes, is useful under conditions in which it is desirable to have attraction of monocytes in tissues and,
concomitantly, inhibition of tissue destruction. These conditions include infectious disorders, in which monocytes can be useful inasmuch as they partecipate in destruction of invading microorganisms but their activation can lead to tissue damage. The present invention is also useful to promote attraction of monocytes at sites of tissue repair and wound healing, where polypeptide growth factors secreted by mononuclear phagocytes promote reconstitution of tissue integrity, but it is desirable to inhibit destructive enzymes produced by the same cells.
Mononuclear phagocytes can kill tumor cells and macrophage infiltration has been associated with tumor regression (for review 10). On the other hand enzymes that digest the extracellular matrix, produced by tumor cells or by other components of the tumor tissue, promote cancer invasion and metastasis (e.g. 11). The present invention is therefore useful to attract monocytes in tumors while inhibiting the enzymes that they utilize to invade and disseminate.
For the intended therapeutic purposes, the CF/TIMP of the invention will be administered in form of suitable pharmaceutical formulations, as described for istance in "Remington's Pharmaceutical Sciences Handbook" Mack Pub. Co., NY, USA, XVII ed. A preferred administration route will be the parenteral route, at dosage ranging from 0.01 to 1000 mg/day. Different administration routes already used for polypeptides such as the topical, nasal, rectal, can also be used according to specific needs the physician may have.
Table 1
Chemotactic activity for monocytes of CF/TIMP
Stimulus Concentration No. of migrated monocytes (in 5 oil fields, ± SD)
Medium 21±3 fMLP 10-8 M 67±2 C5a 5% 154±5 CF/TIMP 1/50 67±3
1/100 40±2
1/200 24±2
Migration across polycarbonate filters was measured in a 90 min. assay. CF/TIMP was HPLC fraction 71 from SW 626 with an estimated protein concentration of 10 mg/ml. The same fraction gave one band in SDS-PAGE (silver stain) and was used for sequencing.
The following examples further illustrate the invention.
EXAMPLE 1 Production of supernatant from SW 626 The human ovarian carcinoma cell line, SW 626, stabilized by Dr. I. Foglhi - Memorial Sloan Kettering Institute, New York, was obtained throught Dr. S. Menard - Istituto Nazionale Tumori, Milano. SW 626 were grown in RPMI 1640 medium, supplemented with 20% fetal bovine serum. In subconfluent cell conditions, the conventional medium was substituted with RPMI 1640 without serum and collected 24 hours later.
EXAMPLE 2
Chemotactic assay
Cell: leucocyte populations were obtained from the buffy coats of blood donations from normal volunteers throught . the courtesy of Centro Trasfusionale, Ospedale Sacco, Milano, Italy. Blood was heparinized (20 μl/ml), diluted one sixth with phosphate-buffered saline (PBS; Gibco Biocult, Glasgow, U.K.) and washed once at 400 g for 10 min. to remove plasma and platelets. Pellets were resuspended in PBS and centrifugate on Ficoll-Hypaque (Lymphoprep; Nyagad, Oslo Norway) at 400 g for 30 min. at room temperature. Peripheral blood mononuclear cells were collected at the interface and were washed twice with PBS, then resuspended at 1 x 10 /ml in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS; GIBCO). To purify PMN, after Ficoll-Hypaque centrifugation, the pellet containing erythrocytes and polymorphonuclear leukocyte (PMM) was resuspended in Hanks'balanced salt solution (HBSS; GIBCO) and mixed with Eufusin (STHOLL Farmaceutici, Modena, Italy) at a concentration of 3 ml of Eufusin per 1 ml of blood cells. The leukocyte rich supernatant obtained after 40 min. of 4βC incubation was then centrifuged, and the cells pellet was mixed with distilled water for 30 sec. to lyse residual erythrocytes. Cells were the washed twice and resuspended at 1 x 10 /ml in HBSS with 0.2% human serum albumin (HSA; Sigma Chemical
Co., St. Louis, MO). The final preparation contained greater than 95% PMN. - Chemotactic agents: N-formylmethionyl-leucyl- phenylalanine (FMLP; Sigma Chemical Co., St. Louis, MO) was used as standard chemotactic agent.
FMLP was dissolved in DMSO at a final
_3 concentration of 10 M and stored at 20βC.
The final concentration used as positive control was 10"8-10"7 M. - Proceedure: migration of monocyte and PNM was evaluated by a microchamber technique. Twenty-five microliters of each sample were seeded in the lower compartment of the chemotaxis chamber, and
50 μl of cells suspension (1 x 10 /ml for each cells type) were seeded in the upper compartment.
The two compartments were separated by a 5 μm pore-size polycarbonate filter (Nucleopore Corp. , Pleasanton. CA) ; and on polyvinylpirrolidone PVP- free for PMN. The chambers were incubated at 37°C in air with 5% C02 for 90 (monocytes) and 120 (PMN) min. At the end of incubation, filters were removed, fixed and stained with Diff-Quik (Harleco, Gibbstown, NJ) , and five oil immersion fields were counted after coding samples. EXAMPLE 3
- Protocol of purification by Silicic acid absorption: the supernatant from SW 626 cell line was concentrated and partially purified by batch adsorption to silicic acid (Mallinckrotd Inc., Paris, KY) at neutral pH for 2 hours at 4βC. After centrifugation at 3000 rpm and washed with
Dulbecco's PBS, biological activity was recovered by sequential elution with 50% ethylene glycol in 1.4 M NaCl.
Heparin-Sepharose column: the eluate from silicic 5 acid was dialyzed against 50 mM Tris, 50 mM NaCl pH 7.4 and loaded (10 ml/h) to a heparin-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) column (0.9 x 15 cm; 10 ml bed volume) equilibrated with the same buffer. After washing with the equilibrating
10 buffer, the column was subjected (20 ml/h, 5 ml fractions) to an NaCl gradient (0-2 M) in 50 mM Tris pH 7.4.
FPLC separation: the active fractions from the heparin-Sepharose column were dialyzed against 50
15 mM formate pH 4.0 and subjected to FPLC, using a Mono-S cation-exchange column (Pharmacia) . After an initial wash with 50 mM formate pH 4.0, the activities were eluted (1 ml/min., 1 ml fractions) with a linear NaCl gradient (0-1 M) in the same
20 buffer.
Absorbance was monitored at 280 nm as a parameter for protein concentration.
HPLC separation: the FPLC fractions containing monocyte chemotactic activity were finally
25 subjected to reversed-phase HPLC on a C8 Aquapore RP-300 column (Applied Biosystems, Foster City CA) in an LKB HPLC system (Bromma, Sweden). The column was equilibrated with 0.05% (by vol.) trifluoroacetic acid and eluted with a linear
30 acetonitrile gradient (0-100% buffer B; buffer B is 80% by vol., acetonitrile in equilibration
buffer). The flow was mantained at 0.4 ml/min. and 1 min. fractions were collected. The column affluent was monitored at 280 nm.
SDS PAGE: the purity of chemotactic activity was 5 tested by SDS-PAGE.
Samples (20 μl) were loaded onto a linear gradient 10-25% (mass/vol.) polyacrylamide gel, containing 0.1% (mass/vol.) SDS, with a 5% stacking gel on top, containing 0.25% (mass/vol.) SDS. The 10 dimensions of the gels were 17 x 13 x 0.1 cm. The molecular mass markers (Bio-Rad, Richmond, CA) used were phosphorylase b (Mr 92500), bovine serum albumin (Mr 66200), ovalbumin (Mr 45000), carbonic anhydrase (Mr 31000), soybean trypsin inhibitor 15 (Mr 21500), lysozy e (Mr 14400), and the low-Mr markers (Pierce chemical Company, Rockford, IL) aprotinin (Mr 6500). Silver staining of the proteins in the gels was carried out.
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