WO1989000164A1

WO1989000164A1 - Chondrocyte-specific membrane proteins and process for diagnosis and monitoring of autoimmune reactions against chondrocytes

Info

Publication number: WO1989000164A1
Application number: PCT/EP1988/000572
Authority: WO
Inventors: Klaus Von Der Mark; Jürgen MOLLENHAUER
Original assignee: Max-Planck-Gesellschaft Zur Förderung Der Wissensc
Priority date: 1987-07-01
Filing date: 1988-06-30
Publication date: 1989-01-12
Also published as: DE3721790A1; DE3721790C2

Abstract

Chondrocyte-specific membrane proteins which display an immune reaction with autoantibodies can be obtained by homogenizing cartilage tissue, isolating the membrane vesicles from the homogenizate, and separating and extracting said proteins from the vesicles. The chondrocyte-specific membrane proteins are suitable for diagnosis and monitoring autoimmune reactions against chondrocytes.

Description

"Cartilage cell-specific membrane proteins and methods for the diagnosis and monitoring of autoimmune reactions against cartilage cells"

The invention relates to the provision of cartilage cell-specific membrane proteins for the detection of immune reactions with autoantibodies.

The invention also relates to methods for diagnosing and monitoring the course of autoimmune reactions against cartilage cells.

Diseases of the cartilage are usually accompanied by autoimmune reactions. The group of these diseases includes, for example, chronic polyarthritis, osteoarthritis or gout. These are some of the most common civilization diseases. They are caused by improper nutrition, occupational damage to posture, but also by intensive sporting activities. In the literature, components of the extracellular matrix of the cartilage, such as collagen type II (NA Andriopoulos et al. (1976), Arthritis Rheum. 19, 613; DC), are the targets of the cellular and humural autoimmune reaction which occur as a concomitant symptom of the diseases mentioned Haynes et al., (1985), J. Rheumatol. 13, 358), histocompatibility antigens (GR Burmester et al. (1984), Rheumatol Int. 4 (supp), 31), nuclear proteins and single-stranded DNA (NA Andriopoulos et al (1976), Arthritis Rheim. 19, 613). However, these antigens are immunologically little or not at all specific for the cartilage cell or, as in the case of the matrix proteins, are not potent antigens. Therefore, they are not suitable test antigens and cannot be used to clearly identify immunoreactivity and clinical picture. Thus, in spite of intensive research work, neither a reliable diagnosis nor a follow-up has been possible for the diseases mentioned so far. The importance of diagnostic methods lies in the targeted selection of suitable medications. The quantitative control of the course of the disease can be used, for example, to control the dose and duration of the application of the selected drugs.

The invention is therefore based on the object of providing effective test antigens which permit diagnosis and monitoring of the diseases mentioned, in particular in humans. Another object of the invention is to provide a method for diagnosing and monitoring the diseases mentioned, in particular those which occur in humans.

These objects are achieved by the invention.

The invention thus relates to cartilage cell-specific membrane proteins which have an immunoreactivity with autoantibodies and are obtainable from

- homogenization of cartilage tissue,

- Isolation of the membrane vesicles from the homogenate; and

- separation and extraction from the vesicles.

The invention also relates to cartilage cell-specific membrane proteins which have immunoreactivity with autoantibodies and are obtainable from

- Homogenization of cartilage tissue, - Isolation of the membrane vesicles by density gradient centrifugation from the homogenate and

Obtaining individual cartilage cell-specific membrane proteins by electrophoresis of the membrane vehicle in an SDS polyacrylamide gel and electrophoretic elution of the proteins from the SDS polyacrylamide gel. Specific examples of cartilage cell-specific membrane proteins of the invention are by relative molecular weights (M) of 155,000, 116,000, 78,000, 76,000, 66,000,

42,000, 38,000, 30,000 and 28,000 respectively.

Another object of the invention is a method for the diagnosis and course control of autoimmune reactions against cartilage cells, which is characterized in that a mixture of cartilage cell-specific membrane components is used as test antigen in an immunological detection method for the detection of autoantibodies.

According to the invention, the expression “cartilage cell-specific membrane component” denotes components of the covering of cartilage cells of vertebrates that do not occur in the nearer or further extracellular cartilage matrix and that are not histocompatibility antigens. According to the invention, proteins of the shell of cartilage cells of vertebrates which do not occur in the nearer or further extracellular cartilage matrix and which are not histocompatibility antigens are referred to as "cartilage cell-specific membrane proteins".

According to the invention, not only can cartilage cell-specific membrane components of human cells be used as a test system for the antibodies from human blood, but heterologous materials can also be used, for example cartilage cell-specific membrane proteins from chickens and rats. The main advantage of using these proteins is that they are always available in sufficient quantities. Therefore, the cartilage cell-specific membrane components and membrane 11 proteins around those found in human hyaline cartilage,

Cattle, pigs, rats or chickens occur and react with the autoantibodies found in sick people in the test methods mentioned.

5 ^" The cartilage cell-specific membrane components and membrane proteins of the chicken are particularly preferred. The expression" laboratory or farm animals "used in the description of the present invention refers to animals such as rats, mice, rabbits, guinea pigs, cattle, pigs, rCC Denoted sheep, goat, horse or chicken.

In general, there are also highly specific antigens on the surface of cells, such as differentiation antigens, which guide the cell to typical performance, for example to

Enable 15 selective adhesion to cartilage matrix proteins. According to the invention, it is therefore assumed that the main goal of autoimmune aggression in the context of diseases of the cartilage is not, as previously assumed, the cell-free connective tissue, but that the highly specific antigens of the

20 cartilage cell itself the main goal. The first contact of the immune system with the cartilage cell takes place via its outer membrane. The constituents of the membrane are thus causally at the top of the reaction cascade in auto-sensitization.

25 The validity of this concept is underlined by the results of experiments with rabbits. In these experiments, the rabbits were immunized with cartilage cell membranes or individual membrane proteins by subcutaneous injection of the antigens. As a result of this immunization,

30 degenerative joint damage. Some of the inflammatory reactions in the knee joint of the hind legs of the test animals were so pronounced that open wounds developed on the outside. Reactions of this magnitude have been reported in the literature on the generation of immune reactions against cartilage

35 components not described. - $ ^"- addition of the test sera were no significant Reak¬ tions revealed constituents against used as antigen cartilage cell-specific Membranbe-- human patients with rheumatoid arthritis or osteoarthritis surprisingly clear reactions of detectable autoantibodies In contrast, the autoantibodies with components of the extracellular matrix. The highly specific immune reactions between autoantibodies and cartilage cell-specific membrane constituents found according to the invention thus clearly stand out from the data, which have so far not been convincing from the prior art mentioned above.

The experimental observations according to the invention open up new possibilities for the diagnosis and monitoring of the course of acute and chronic diseases of the cartilage, in particular of chronic polyarthritis ^" and osteoarthrosis. With the methods according to the invention, for example, early diagnosis of arthrotic processes is possible, which is already possible with slight, for example sports-related, damage of the joints occur and are accompanied by significant autoantibody titers.

In addition, the methods according to the invention now make it possible to monitor the course of rheumatoid diseases. Suitable therapeutic measures can thus be selected and controlled. In particular, the validity of medicamentous treatment, for example with chondroprotectives, can be assessed better than hitherto.

The studies carried out according to the invention relate on the one hand to the qualitative detection of autoimmune aggression by circulating antibodies of the patients with the associated specific antigens and on the other hand to the quantitative check whether the titer of these antibodies is correlated with the extent of the disease. \ -

The drawing shows:

1 shows the immunoreactivity in the immunodot assay of sera from six osteoarthritis patients, each titrated in steps of 2 from top to bottom, against cartilage cell membranes, soluble cartilage cell proteins and matrix components from the rat. 0.005 mg of membrane or soluble protein and 0.01 mg of matrix protein were applied to each dot. ιτo: -

Fig. 2 shows the inhibition of autoantibody activity of osteoarthritis with cartilage cell plasma membranes or a

Mixture of different collagens from human cartilage, detected in an ELISA (enzyme linked immunosorbent assay).

15 The low inhibibility of the reaction by collagen indicates the low relevance of these antigens in the measurement of the autoimmune reaction.

3 shows the immune response of two sera (o - control serum,

20 ^" - chronic polyarthritis) in an ELISA, titrated in two steps, in direct comparison of the reactivity of cartilage cell membranes and fibroblast membranes as reactive antigens on the solid phase. By extrapolation of the linear range of the titration curve

25 ^' ve towards the intersection with the y-axis, the intersection values (arrowheads) can be used as a measure of the immunoreactivity. These values are shown collectively in FIG. 4.

acr

35 4 shows the cumulative recording of the extrapolation values from the titration curves of the ELISA (see FIG. 3). A group of polyarthritic sera (•), osteoarthritic sera (A) and control sera (o) with their values for cartilage cell (A), fibroblast (B) and

Pancreatic tumor cell membranes (C) as antigens on the solid phase.

5 shows the mean function with standard deviation of a series of ELISA titrations of osteoarthritic sera

(-) and control sera (). As an antigen on the

Solid phase, 0.005 mg cartilage cell membrane protein was used per 96 well of the microtiter plate from juvenile human cartilage.

6 shows the immunoblot (= Western blot) of a number of osteoarthritis sera. Chicken cartilage cell membrane proteins served as antigen. A = blots; B = Coomassie blue - colored 5-15% SDS-polyacrylamide gradient gel of the membrane proteins, which served as a template for the blot. Column "Mr" = relative molar mass of the additionally applied molar mass marker proteins, expressed in kilodaltons.

7 shows the immunoblot of three polyarthritis sera. Chicken cartilage cell membrane proteins served as the antigen. A = blots;

B = colored blot template (for description see FIG. 6).

8 shows the immunoblot of pooled polyarthritic sera (CP) and osteoarthritic sera (OA) on electrophoretically separated plasma membranes of human pancreatic tumor cells (A) and skin fibroblasts (B). Gel = Coomassie blue-colored reference gel strip from the blot template (see FIG. 6). material and methods

Goat anti-human polyvalent immunoglobulin peroxidase (A 8400), rabbit anti-human serum (H 3383) and 4-chloro-naphtol (C 8890) are obtained from Sigma-Chemie, Munich; Goat anti-rabbit immunoglobulin peroxidase (8940) from Research Plus Laboratories, Denville, NJ, USA; Nitrocellulose paper (0.45 μm. Pore size) from Sartorius GmbH, Göttinqen; 2,2 '.Azino-di-3-ethylbenzthiazoline sulfonate (6) (ABTS, 102956) from Boehrinqer, Mannheim. All other chemicals were purchased in p.A. quality from Merck, Darmstadt.

Preparation of plasma membranes

Plasma membranes from cell culture (human foreskin fibroblasts, ductal pancreatic adenocarcinoma): The cells are scraped off the culture vessel and in 8.5% (w / w) sucrose, 0.01 M Tris HC1, pH 7.5, 1 mM protease inhibitors (N -Ethylmalein- imid, phenylmethylsulfonic acid) transferred. The further preparation is carried out according to Cates and Holland (1978), 40%, 17% and 8.5% sucrose (w / w), the latter with the sample, being present in the sucrose gradient from bottom to top. The plasma membranes are concentrated on the 40% / 17% boundary layer. After dialysis against phosphate buffered saline (PBS) + protease inhibitors. the membranes are portioned and frozen.

Membranes of human, rat and chicken cartilage, human articular cartilage from surgical material, rat or chicken breast cartilage are homogenized with an Ultraturrax in 8.5% sucrose buffer as described above. The crushed solid becomes by centrifugation at 1500 g, 15 min., 4 ° C

Material separated from the supernatant, which contains the membranes and soluble proteins. The procedure is repeated with the solid material and the supernatants are combined. All vesicles are pelleted from this supernatant by a further centrifugation (100,000 g, 90 min., 4 ° C.). The pellet is resuspended in a small volume of 8.5% sucrose buffer and ~ 3 - standing centrifuged in sucrose gradient. The 40% / 17% boundary layer contains the cartilage cell membranes.

Immunodot assays of 0.002 mg membrane protein are drawn onto the nitrocellulose per well of the incubation chamber (biorad). The paper is then saturated with 3% skim milk powder in PBS for 2 hours. The sera to be examined are titrated in steps of 2 or 3 in PBS / milk powder and incubated on the plate for 2 hours. After washing five times with PBS, the reaction is carried out with the detection antibodies. This can be indirect or direct, the indirect one being approximately 10 times more sensitive. In the first case (indirectly) the paper is incubated with rabbit anti-human IgH diluted 1: 500 (PBS / milk powder) for 2 hours, washed five times with PBS and a further 2 hours with goat anti-rabbit diluted 1: 200 Incubated peroxidase. After washing ten times with PBS, the detection reaction is carried out with chloronaphtol (dilute stock solution (10 mg chloronaphtol in 10 ml methanol) 1: 5 in PBS, pH 6.5, and add 100 μl HO- to 100 ml). The reaction should take place in the dark, since the resulting dye is sensitive to light. The titration of the sera can now be read directly from the blue color or measured in a reflection photometer. ι _m th case of the direct detection method is by incubation with the Patien¬ tenseren directly goat anti-human-peroxidase (1: 500) was applied otherwise as described above.

ELISA (after E.Engvall, P. Perlmann (1971) Immunochemistry 8, 871)

For optimal adhesion of the antigens on the ELISA plate, 0.002 mg membrane protein in 100 μl HO deion are used. suspended, applied per hole and adsorbed onto the plate by freeze-drying. The plate is now saturated with skimmed milk powder (3%, PBS) for 2 hours and can be used as described above for the immunodot. As a color reaction - lo - here serves ABTS (according to B. Porstmann et al., J. Clin. Chem. Clin. Biochem 19 (1981), p. 435), the color values are read in the ELISA reader (Dynatech MR 700).

Immunobiot (after H. Towbin, T. Shoehdin, J. Gordon (1979), Proc. Natl. Acad. Sei. USA 76, 4350)

The membrane proteins are separated in SDS gel electrophoresis (according to UK Laemmli, Nature 227 (1970), p. 680) in a 5-15% polyacrylamide gel after reduction with dithiothreitol. Typically 0.1 mg membrane protein is applied per gel lane. A reference gel strip is stained with Coomassie blue, the rest is transferred electrophoretically to a mitrocellulose paper overnight at room temperature. The electrophoresis buffer contains 0.1% SDS. After the transfer has been completed, the paper is saturated with milk powder / PBS and incubated with the individual patient sera in suitable dilution (PBS / milk powder solution) overnight at 4 ° C. Bound immune globulins be makes above ^'for the immunodot beschrie¬ ben in the direct or indirect detection methods visible ge, wherein the individual incubation with neighboring facing antibodies take place overnight at 4 ° C.

The examples illustrate the invention.

Example 1

Comparison of the anti-qenity of different cartilage fractions and cartilage of different species

First, it was investigated how the immune reactivity of cartilage matrix components, soluble components and membranes is in direct comparison. For this purpose, 0.01 mg each of these proteins was applied in the immunodotassay and incubated with the sera of a number of arthrotic patients. 1 shows the reactivity of a typical serum with the respective fractions from rat cartilage. The surprising result is the strongest - U _

activity of the membrane fraction, together with the very weak, hardly detectable reactivity of the sera on matrix protein. The inhibition ELISA further underlines the reduced immunoreactivity of extracellular matrix proteins compared to the plasma membranes (FIG. 2). The antibody activity in the patient's serum can be completely inhibited with plasma membranes, while even by combining all types of collagen of the cartilage only about 30% inhibition of the sera can be achieved.

Example: 2

Titer determination of various patient sera on chicken and human cartilage cell membranes

3 and 4 show the titration of patient and control sera in the ELISA on chicken cartilage membrane as an antigen. 5 shows the cross-reactivity of some sera against human fibroblast membranes and against membranes from a pancreatic adenocarcinoma cell line. The reaction against cartilage cell membranes in the case of sera from polyarthritics as a whole, at least in some cases with arthrotics, is clearly above the values of the control groups. On the other hand, there is only a non-specific reactivity in the control area on the fibroblast and tumor cell membranes. Irrespective of these experiments, the ELISA titrations of osteoarthritis patient sera and controls shown in FIG. 5 were carried out with juvenile human cartilage as the antigen. They show the same trends as the experiments shown above, but are statistically significant due to the larger number of subjects on which the experiments are based. It is crucial in the findings shown in Examples 1 and 2 that the same results were achieved with the aid of three different assay systems (immunodot, ELISA and inhibition ELISA).

As a qualitative aspect, it is very clear that the cell membranes appear as the dominant target of the autoimmune reaction, whereas the components examined by other groups are of little importance.

The comparison of the quantitative data shows that diseases of the cartilage can be detected with the help of these serological test methods. In particular, the significant titer differences between the three groups of subjects examined (control, osteoarthritis as a local disease and chronic polyarthritis as a serious systemic disease) show that quantitative quantitative differences in the clinical picture can be detected with objective measurement parameters, which was previously not possible .

The proven immunoreactivity of the animal membrane components of the cartilage with human sera is of considerable importance for the commercial use of the test system, since human cartilage is not available to an unlimited extent as test antigen.

- -

- Example 3

Representation of the immunoreactive individual components from the cartilage cell membranes

5

The decisive factor for the quality of the antibody detection system is its discriminatory property. In the present case that only a defined number of proteins from the whole of the cartilage cell specific membrane protein ₀ ^m is expected to respond it the patient sera.

6 and 7 show immunoblots with some sera from patients with osteoarthritis (FIG. 6) or chronic polyarthriti (FIG. 7). 8 shows a control immunobiot with combined sera from osteoarthritis and chronic polyarthritis patients and with human fibroblast and pancreatic tumor cell membranes as the 'antigen. The defined areas in the blots in FIGS. 6 and 7 and their absence in FIG. 8 are to be noted. This is a strong indication of the cartilage cell specificity of the immune reaction.

The recognized cartilage cell antigens are listed in Table I according to their relative molecular weight. Thereby osteoarthritis and chronic polyarthritis are compared directly. It can be seen that the antigens are essentially identical. This is a further indication of the high specificity of the proven immune response.

0

5 - IH -

Table I

Comparison of the reactive antigens of osteoarthritis and chronic polyarthritis autoantibodies in the body

Western blot.

Mr osteoarthritis chron. Polyarthritis

155,000 ++

135,000 ++

116,000 ++

78,000 ++

76,000 ++

66,000 ++

42,000 +

38,000 +

30,000 +

28,000

Example 4

Inhibition ELISA with different cartilage cell membranes to show the specificity of the immune response

As in Example 3, a statement should also be made here about the discriminatory properties of the detection system, here of the ELISA. With chicken cartilage cell membranes as detection antigen, it is checked to what extent different types of membrane in suspension can inhibit the reactivity of the patient sera.

Table II shows an overview of the results obtained. It can be seen that membranes from chicken cartilage can inhibit the activity of polyarthritic sera, so that the residual activity corresponds to the background reaction of the control sera. Membranes from rat and human cartilage cells inhibit similarly well. When the membranes are treated with enzymes that digest the extracellular matrix from the surface of the membrane vesicles (collagenase, hyaluronic acid, chondroitinase ABC), the inhibitory capacity is also retained. Participation of collagen and proteoglycans in a (possibly unspecific secondary) immune reaction can therefore be excluded. In contrast, the non-specific protease trypsin completely destroys the cell surface proteins and thereby eliminates any inhibitory activity of the membranes treated in this way. The digested peptides released by trypsin can also no longer bind to the body. In analogy to the negative reaction of the patient sera to fibroblast and pancreatic tumor cell membranes documented in Example 2, these membrane types are also not able to block the immune reaction of the patient sera. - U -

Table II Inhibition of Antibody Activity in Chronic Polyarthritis (C.P.) Sera by Plasma Membranes.

Percentage of uninhibited C.P. sera (median + standard deviation)

C.P. sera control sera

Inhibitor

None 100 27.4 ± 5.9

Chicken M. 31.9 ± 7.0 17.1 ± 3.2

"Matrix free" 40.6 ± 1.4 18.3 + 0.0 "Trypsinized" 102 ± 55 33.0 ± 0.6 "Trypt. Peptides" 100 ± 19.2 31.9 ± 3.5

Rats-M. 61.6 ± 4.3 19.6 ± 7.0 Human M. 57.4 ± 5.6 17.7 ± 8.6

HFF 93.3 * ^• nd PaTuII 84.1 ± 9.1 25.9 + 11.6

Explanation: Chicken M , Rat-M. , Human-M. = Cartilage line plasma membranes of the respective species; Chicken-M treated with "matrix-free" = Si multan with collagenase, chondroitinase ABC and hyaluronidase. ; "Trypsinized" = trypsin-treated (4h, 37 ^β C) chicken M .; "Trypt. Pept." = tryptic peptides formed during the trypsin treatment; HFF = human foreskin fibroblast membrane; PaTu II = human pancreatic carcinoma cell membrane; * = Individual measurement; nd = not carried out.

These experiments show that only the presence of intact cartilage cell surface proteins in the reaction mixture can block the reaction of the sera in the ELISA, almost completely. A non-specific adsorption reaction of the polyarthritic sera on the ELISA substrate can therefore be excluded.

Claims

- /? - “Cartilage cell-specific membrane proteins and methods for diagnosing and monitoring the course of autoimmune reactions against cartilage cells” patent claims

1. Cartilage cell-specific membrane proteins that have immune reactivity with autoantibodies and are available through

- Homogenization of cartilage tissue;

- Isolation of the membrane vesicles from the homogenate,

- Separation and recovery from the vesicles.

2. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has a relative molecular weight (M _r ) of 155,000.

3. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 116,000.

4. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an _r of 78,000.

5. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M of 76,000.

6. Cartilage cell-specific membrane protein according to claim 1, characterized in that it is an M^. of 66,000.

^"~ . Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 42,000.

8. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 38,000.

9. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 30,000.

10. Cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 28,000.

11. Cartilage cell-specific membrane protein according to one of claims 1 to 10, characterized in that it is available from human cartilage cells or from cartilage cells from laboratory or farm animals.

12. Cartilage cell-specific membrane protein according to one of claims 1 to 10, characterized in that it is available from chicken cartilage cells.

13. Cartilage cell-specific membrane proteins that have immune reactivity with autoantibodies and are available through

- homogenization of cartilage tissue,

- Isolation of the membrane vesicles from the homogenate by density gradient centrifugation; and

- Obtaining individual ^cartilage cell ^membrane proteins by electrophoresis of the membrane vesicles in an SDS-polyacrylamide gel and electrophoretic elution of the proteins from the SDS-polyacrylamide gel.

14. Method for obtaining cartilage cell-specific

Membrane proteins that have immunoreactivity with autoantibodies, characterized in that cartilage tissue is homogenized, the membrane vesicles are separated from the homogenate by density gradient centrifugation and individual cartilage cell-specific membrane proteins are isolated by electrophoresis of the membrane vesicles in an SDS-polyacrylamide gel and electrophoretic elution of the Proteins are obtained from the SDS-polyacrylamide gel.

15. Method for diagnosing and monitoring the course of autoimmune reactions against cartilage cells, characterized in that a mixture of cartilage cell-specific membrane components is used as a test antigen in an immunological detection method for detecting autoantibodies.

16. The method according to claim 15, characterized in that a single cartilage cell-specific membrane component is used as a test antigen.

17. The method according to claim 15 or 16, characterized in that a protein according to one of claims 1 to 11 is used as the cartilage cell-specific membrane component.

13. The method according to any one of claims 15 to 17, characterized in that the immunological detection method is an immunodot assay, an immunoblot assay, an ELISA, or an inhibition ELISA.

19. The method according to any one of claims 15 to 18, characterized in that when using plastic reaction vessels, such as microtiter plates, the test antigen is adsorbed onto the surface of the plastic reaction vessel by freeze-drying. - 2θ -

20. Cutlery for diagnosing and monitoring the course of autoimmune reactions against cartilage cells, containing at least one cartilage cell-specific membrane protein according to one of claims 1 to 13 and usual immunological detection agents.