DE3721790C2 - Cartilage cell-specific membrane proteins and cutlery for the diagnosis and monitoring of autoimmune reactions against cartilage cells - Google Patents

Description

The invention relates to the provision of cartilage cell speci fish membrane proteins for the detection of immune reactions Autoantibodies.

The invention also relates to cutlery for diagnosis and Monitoring of autoimmune reactions against cartilage cells.

Cartilage disorders are usually caused by autoimmune reactions accompanied. Belong to the group of these diseases for example chronic polyarthritis osteoarthritis or Gout. These are some of the most common emerging civilization diseases. You will emerge due to improper nutrition, occupational damage to posture, but also through intensive sporting activities.

As goals of as a side effect of the mentioned crane cellular and humoral autoimmune cracks tion are components of the extracellular in the literature Cartilage matrix like collagen type II (N.A. Andriopoulos et al. (1976) Arthritis Rheum. 19, 613; D.C. Haynes et al., (1985), J. Rheumatol. 13, 358), histocompatibility antigens (G.R. Burmester et al. (1984), Rheumatol Int. 4 (supp), 31), Nuclear proteins and single-stranded DNA (N.A. Andriopoulos et al. (1976), Arthritis Rheim. 19, 613). These However, antigens are immunologically little to no specific fish for the cartilage cell or else no potent antigens as well as for matrix proteins, as described in EP-A 1 145 681 it is not clearly clarified whether they are potent antigens. Therefore, they are not suitable test antigens and cannot be used for a clear Assignment of immunoreactivity and clinical picture used will.  

So far was with the mentioned diseases despite research is neither a reliable diagnosis a progress check is still possible. The importance of diag nostic process lies in the targeted selection of suitable medication. With quantitative control of the patient For example, the dose and duration of the Control the application of the selected medication.

The invention is therefore based on the object of being effective Test antigens provide a diagnosis and history control of the diseases mentioned, especially in men allow, allow.

These objects are achieved by the invention.

The invention also relates to cartilage cell-specific Membrane proteins that have immunoreactivity with autoantibodies have pern and are available through

• - homogenization of cartilage tissue,
• - Isolation of the membrane vesicles by density gradients centrifugation from the homogenate and
• - Obtaining individual cartilage cell-specific membranes protein by electrophoresis of membrane vehicles in one 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 characterized by relative molecular weights (M _r ) of 155,000, 116,000, 78,000, 76,000, 66,000, 42,000, 38,000, 30,000 and 28,000, respectively.

The invention also provides cutlery for Diagnosis and monitoring of autoimmune reactions against Cartilage cells, the test antigen is at least one cartilage cell-specific Contain membrane protein and optionally usual immonological detection agents.

The expression "cartilage cell-specific membrane component" denotes components of the envelope of Knor according to the invention fur cells from vertebrates that are not in the near or further extracellular cartilage matrix occur and at which are not histocompatibility antigens. According to the invention, proteins are the shell of cartilage cells of vertebrates that are not near or far extracellular cartilage matrix occur and the no histo compatibility antigens are called "cartilage cell specific Membrane proteins ".

According to the invention can be used as a test system for the anti body from human blood not only cartilage cell specifi use membrane components of human cells, but heterologous materials can also be used the, for example cartilage cell-specific membrane proteins of chickens and rats. The use of these proteins has the main advantage that they are always in sufficient large quantities are available.

Therefore, it can be used as test antigens cartilage cell-specific membrane components and membrane  proteins around those found in human hyaline cartilage, Beef, pork, rat or chicken occur and with those in it sick autoantibodies occurring in the ge named test procedures react.

The cartilage cell-specific memes are particularly preferred Chicken industry components and membrane proteins.

With the ver in describing the present invention The term "laboratory or farm animals" is used for animals such as Rat, mouse, rabbit, guinea pig, beef, pork, Denoted sheep, goat, horse or chicken.

Generally there are on the surface of cells also highly specific antigens, such as differentiation antigens, which the cell at typical performance, for example Enable 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, as previously assumed, not the cell-free bandage tissue, but that the highly specific antigens of Cartilage cell itself is the main goal. The first con the immune system with the cartilage cell takes place over their outer membrane instead. So that the constituent le of the membrane causally in the first place of the reaction cascade in auto sensitization.

The validity of this concept is underlined by the Results of experiments with rabbits. With these Experi rabbits with cartilage cell membranes or individual membrane proteins by subcutaneous injection of the Immunized antigens. As a result of this immunization develop they had degenerative joint damage. Partially the Ent ignition reactions in the knee joint of the hind legs of the ver search animals so pronounced that wounds open to the outside developed. Reactions of this magnitude are in the litera tur about the generation of immune reactions against cartilage components not described.  

The test also revealed sera from human patients chronic polyarthritis or osteoarthritis surprising clear reactions of the detectable autoantibodies against the cartilage cell-specific membrane used as antigen components. In contrast, no clear reak tions of autoantibodies with components of the extracellular Matrix or found with soluble proteins.

The highly specific immunreak found according to the invention between autoantibodies and cartilage cell-specific Membrane components stand out clearly from to little known from the above-mentioned prior art convincing data.

The experimental observations according to the invention open up new possibilities for diagnosis and follow-up acute and chronic diseases of the cartilage, in particular re of chronic polyarthritis and osteoarthrosis. With the cutlery according to the invention, for example Early diagnosis of arthrotic processes possible that are already at slight, for example sports-related damage to the Ge steer occur and of significant autoantibody titers to be accompanied.

The cutlery according to the invention also enables now the follow-up control of rheumatoid diseases. There suitable therapeutic measures can be selected with being controlled. In particular, the validity can be medicinal töser treatment, for example with chondroprotectives, esp be assessed more than before.

The investigations carried out with the cutlery according to the invention relate to on the one hand, the qualitative evidence of autoimmune aggression by circulating antibodies of the patients with the specific antigens and on the other hand the quan tive check whether the titer of these antibodies with the extent the disease is correlated.  

The drawing shows:

Fig. 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.

Fig. 2 shows the inhibition of autoantibody activity of Osteoarthritisseren with cartilage cell plasma membranes or a mixture of different collagens from human cartilage, as demonstrated in the ELISA (enzyme linked immunosorbent assay). The low inhibibility of the reaction by collagen indicates the low relevance of these antigens in the measurement of the autoimmune reaction.

Fig. 3 shows the immune response of two sera (○ - control serum, ⚫ - chronic polyarthritis) in the 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 extrapolating the linear range of the titration curve ve to 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.

Fig. 4 shows the cumulative detection of the extrapolation values of the titration curves of the ELISA (see Fig. 3). A group of polyarthritic sera (⚫), osteoarthritic sera (∆) and control sera (○) with their values on cartilage cell (A), fibroblast (B) and pancreatic tumor cell membranes (C) as antigens on the solid phase are recorded.

Figure 5 shows the mean value function with standard deviation of a series of ELISA titrations of Osteoarthritisseren. (-) and the controls (---). 0.005 mg of cartilage cell membrane protein per 96 well of the microtiter plate from juvenile human cartilage was used as the antigen on the solid phase.

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

Fig. 7 shows the immunoblot of three Polyarthritisseren. Chicken cartilage cell membrane proteins served as the antigen. A = blots; B = colored blot template (for description see FIG. 6).

Fig. 8 shows the immunoblot pooled Polyarthritisseren (CP) and Osteoarthritisseren (OA) on electrophoretically sepa-configured plasma membranes of human Pankreastumorzel len (A) and dermal fibroblasts (B). Gel = Coomassie blue colored reference gel strip of 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 para pier (0.45 µm pore size) from Sartorius GmbH, Göttingen; 2,2′-azino-di-3-ethyl-benzothiazoline sulfonate (6) (ABTS, 102956) by Boehringer, Mannheim. All other chemicals were in p.A. quality sourced from Merck, Darmstadt.

Preparation of plasma membranes

Plasma membranes from cell culture (human foreskin fibro blasts, ductal pancreatic adenocarcinoma): the cells scraped from the culture vessel and in 8.5% (w / w) sucrose, 0.01 M Tris HCl, pH 7.5, 1 mM protease inhibitors (N-ethylmalein imide, phenylmethylsulfonic acid). The further prep ration takes place according to Cates and Holland (1978), whereby in Sucrose gradients from bottom to top 40%, 17% and 8.5% sucro se (w / w), the latter with the sample. The plasmamem branches are concentrated at the 40% / 17% boundary layer. To Dialysis against phosphate buffered saline (PBS) + proteaseinhi Bitter the membranes are portioned and frozen.

Membranes of human, rat and chicken cartilage, human joint cartilage made of surgical material, rat or chicken breast Cartilage is made with an Ultraturrax in 8.5% sucrose buffer homogenized as described above. Through a centrifuge gation at 1500 g, 15 min., 4 ° C the crushed solid Material from the supernatant containing the membranes and soluble protein contains, separated. The procedure is with the Festmate rial repeated and the supernatants combined. Through a white tere centrifugation (100,000 g, 90 min., 4 ° C) are all Ve pellet pelleted from this supernatant. The pellet is in one small volume 8.5% sucrose buffer and resuspended as before  described centrifuged in a sucrose gradient. The The 40% / 17% boundary layer contains the cartilage cell membranes.

Immunodot assay

Each 0.002 mg of membrane protein per well of incubation chamber (biorad) mounted on the nitrocellulose. The paper is then with 3% skimmed milk powder in PBS for 2 hours saturated. The sera to be examined are in 2 or Titrated in steps of 3 in PBS / milk powder and opened for 2 hours incubated the plate. After washing five times with PBS carried out the reaction with the detection antibodies. These can be indirect or direct, with the indirect one et wa has 10 times higher sensitivity. In the first case (indirectly) the paper is diluted with 1: 500 (PBS / milk powder) rabbit anti-human IgH incubated for 2 hours, five times washed with PBS and diluted 1: 200 for a further 2 hours Goat anti-rabbit peroxidase incubated. After washing ten times the detection reaction with chloronaphtol is carried out with PBS leads (stock solution (10 mg chloronaphtol in 10 ml methanol) Dilute 1: 5 in PBS, pH 6.5, and add 10 µl H₂O₂ to 100 ml ben). The reaction should proceed in the dark as the ent standing dye is sensitive to light. The titration of the Seren can now be read directly from the blue color or be measured in the reflection photometer. In the case of direct The detection method is used after the patient has been incubated tenseren directly applied goat anti-human peroxidase (1: 500), otherwise the procedure is as described above.

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

For optimal adhesion of the antigens on the ELISA plate, who the 0.002 mg membrane protein, in 100 ul H₂O deion. suspended, applied per hole and freeze-dried on the plate adsorbed. The plate is now covered with skimmed milk powder (3%, PBS) saturated for 2 hours and can be used as above for described the immunodot be used. As a color reaction  here serves ABTS (according to B. Porstmann et al., J. Clin. Chem. Clin. Biochem 19 (1981), p. 435), the color values are checked in the EMSA reader (Dynatech MR 700).

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

The membrane proteins are analyzed in SDS gel electrophoresis (according to U.K. Laemmli, Nature 227 (1970), p. 680) in a 5-15% polyacrylamide gel according to Re Production separated with dithiothreitol. Typically who the 0.1 mg membrane protein per gel track. A refe Renzgelstreifen is colored with Coomassie blue, the rest is electrophoretically transferred onto a mitrocellulose paper Transfer night at room temperature. The contains Electrophoresis buffer 0.1% SDS. After the transfer the paper is saturated with milk powder / PBS and with the individual patient sera in appropriate dilution (PBS / milk powder solution) incubated overnight at 4 ° C. Bound immune globulins are described for the immunodot as above visible in direct or indirect verification makes, the individual incubation steps with the Nach antibodies take place overnight at 4 ° C.

The examples illustrate the invention.

example 1 Comparison of the antigenicity of different cartilage fractions and Cartilage of different species

First, it was examined how the immunoreactivity of cartilage matrix components, soluble components and membranes is compared directly. 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. Fig. 1 shows the reactivity of a typical serum with the respective fractions from rat cartilage. The surprising result is the strongest reactivity 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

In Fig. 3 and 4, the titration of patient and control sera in ELISA for chicken cartilage membrane as antigen Darge provides. In FIG. 5, the cross-reactivity shown eini ger sera against human fibroblast membranes and membranes to a Pankreasadenokarzinomzellinie. The reaction against cartilage cell membranes lies with sera from poly art critics overall, with arthrotics at least z. T. significantly 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.

Regardless of these experiments, the ELISA titrations of osteoarthritis of the patient serum 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.

Crucial for the findings shown in example 1 and 2 is that using three different assay systems (Immunodot, ELISA and Inhibition ELISA) the same results were achieved.

As a qualitative aspect, it is very clear that the cell membranes as the dominant target of the autoimmune reaction occur, whereas those examined by other groups Components are of minor importance.

The comparison of the quantitative data shows that diseases of the cartilage using these serological test procedures can be demonstrated. Especially the significant ones Differences in titer between the three examined subjects groups (control, osteoarthritis as a local disease and chronic polyarthritis as a serious systemic disease) show that with this test quantitative differences in Disease pattern can be detected with objective measurement parameters what was previously not possible.

The proven immunoreactivity of the animal membrane Components of the cartilage with human sera is significant liche importance for the commercial use of the test system, since human cartilage as test antigen is not in unlimited order are available.  

Example 3 Representation of the immunoreactive individual components from the Cartilage cell membranes

Crucial for the quality of the antibody detection system is its discriminatory quality. In the present case it is expected that only a defined number of proteins from all of the cartilage cell-specific membrane proteins reacted with the patient sera.

FIGS. 6 and 7 show immunoblots with some sera for treatment of patients with osteoarthritis or rheumatoid arthritis th (Fig. 6) (Fig. 7). Fig. 8 shows a control immunoblot with pooled sera from osteoarthritis and chronic polyarthritis patients and with human fibroblast and pancreatic tumor cell membranes as antigen. Note the defined areas in the blots in FIGS. 6 and 7 and their absence in FIG. 8. This is a strong indication of the cartilage cell specificity of the immune response.

In Table I, the cartilage cell antigens recognized are after their relative molecular weight listed. In doing so Directly compare osteoarthritis and chronic polyarthritis chen. It can be seen that the antigens are essentially are identical. This is another indication of the high Specificity of the proven immune response.  

Table I

Comparison of the reactive antigens of osteoarthritis and chronic polyarthritis autoantibodies in the Western blot.

Claims (14)

1. Cartilage cell-specific membrane proteins which have an 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 vesicles in an SDS polyacrylamide gel and electrophoretic elution of the proteins from the SDS polyacrylamide gel.

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 M _r of 78,000. 5. cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 76,000. 6. cartilage cell-specific membrane protein according to claim 1, characterized in that it has an M _r of 66,000. 7. 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 the claims 1 to 10, characterized in that it is available is from human cartilage cells or from cartilage cells of laboratory or farm animals. 12. Cartilage cell-specific membrane protein according to one of the claims 1 to 10, characterized in that it is available is made from chicken cartilage cells.   13. Process for obtaining cartilage cell-specific Membrane proteins that show immunoreactivity with autoantibodies have, characterized, that homogenizing cartilage tissue, the membrane vesicles by density gradient centrifugation from the homogenate separates and individual cartilage cell-specific membrane proteins by electrophoresis of the membrane vesicles in an SDS polyacrylamide gel and electrophoretic elution the protein is extracted from the SDS polyacrylamide gel.   14. Cutlery for diagnosis and monitoring of autoimmune reactions against cartilage cells containing at least a cartilage cell-specific membrane protein according to one of the claims 1 to 12 and common immunological detection agents.