DE19643093C2 - Use of FasL or CD4 · + · / FasL · - · / TH1 cell lines transfected with FasL or of FasL in combination with one or more cytokines for combating TH1 / TH2 diseases - Google Patents

Description

The invention relates to the use of FasL for the treatment of TH1 / TH2 diseases, in particular Leishmaniasen or Listeriosen, and the use of CD4 ⁺ / FasL ^- / TH1 cell lines transfected with FasL for combating THl / TH2 diseases, a composition of matter of FasL in combination with one or more cytokine (s) and their use as medicines, in particular for combating TH1 / TH2 diseases.

TH1 / TH2 diseases are understood to be a spectrum of immunological conditional disorders that are based on a shift in the immune response or inadequate immune response after infection by pathogens such. B. Viruses, bacteria or protozoa.

The immune system of mammals or humans responds to the infection with pathogens by a cellular and / or a humoral immune response depending on the specific characteristics of the pathogen. While the cellular immune response is mostly used against intracellular parasites, the humoral immune response is characterized by the production of antibodies that neutralize the pathogen extracellularly. B lymphocytes are particularly involved in the humoral immune response, while the class of T lymphocytes interferes with both the cellular and the humoral immune response. A distinction is therefore made between two different subclasses of T lymphocytes, namely CD8 ⁺ cells, which carry a surface molecule called CD8, and analogously a subclass (CD4 ⁺ cells), which have the CD4 marker. These CD4-bearing T cells are also referred to as T helper cells (TH).

T helper cells are again divided into two subclasses, the so-called TH1 cells or the TH2 cells. Both cell classes take each central positions in the fight against intra- or extracellular pathogens a. The inflammatory TH1 cells serve the immune response against intracellular pathogens such as Leishmania, viruses or intracellularly parasitic Bacteria, such as listeria. In contrast, TH2 cells activate B lymphocytes, which - as mentioned above - have humoral immunity to Control extracellular pathogens.

It is crucial for an adequate immune response to the infection Meaning that, depending on the type of pathogen, the TH1- or TH2-led immune response prevails. Dominates when infected with intracellular parasitic pathogens the TH2-directed immune response, d. H. there is insufficient or insufficient TH1-directed immune response, for the purposes of this invention there is a TH1 / PTH2 Disease before.

It is known from the literature that TH1 cells have one of the Fas ligands (FasL) or Fas receptor (FasR) controlled path a cytolytic effect unfold (Ashany, D., Song, X., Lacy, E., Nikoliczugic, J., Friedman, S. M., and Elkon, K. B .; Proc. Natl. Acad. Sci. USA 92, 11225-11229, 1995; Ju, S. T., Cui,  H., Panka, D.J., Ettinger, R., and Marshak, R. A .; Proc. Natl. Acad. Sci. L3SA 91, 4185-4189, 1994; Richardson, B.C., Buckmaster, T., Keren, D.F. and Johnson, K.J .; Eur. J. Immunol. 23, 1450-1455, 1993). It was beyond that described that cloned TH1 cells have a high expression rate of FasL im In contrast to cloned TH2 cells (Hahn, S., Stalder, T., Wernli, M., Burgin, D., Tschopp, J., Nagata, S., and Erb, P., Eur. J. Immunol. 25, 2679-2685, 1995; Ramsdell, F., Seaman, M.S., Miller, R.E., Picha, K.S, Kennedy, M.K. and Lynch, D., Int. Immunol. 6, 1545-1553, 1994; Suda, T., Okazaki, T., Naito, Y., Yokota, T., Arai, N., Ozaki, S., Nakao, K. and Nagata, S., J. Immunol. 154, 3806 -3813, 1995). If the FasL binds to its FasR receptor, a cascade of Triggered signals that eventually lead to cell death (apoptosis) (Nagata, S. and Golstein, P., Science 267, 1449-1456, 1995). With the triggering of death one infected cell is also the cycle of multiplication of the intracellular pathogen interrupted.

It is also described that the ability of the inflammatory TH1- Deliver cells, FasL directed, various target cells of different tissues, including macrophages, based on FasR expression in vitro can be killed (Ashany, D., Song, X., Lacy, E., Nikoliczugic, J., Friedman, S.M., and Elkon, K.B; Proc. Natl. Acad. Sci USA 92, 11225- 11229, 1995, Ju, S.T., Cui, H., Panka, D.J., Ettinger, R., and Marshak, R.A .; Proc. Natl. Acad. Sci. USA 91, 4185-4189, 1994; Richardson, B.C., Buckmaster, T., Keren, D.F. and Johnson, K.J .; Eur. J. Immunol. 23, 1450-1455, 1993).

Leishmania is a characteristic example of that class of pathogens that have one cause intracellular infection.

Leishmania (L.) belongs to the Trypanosomatidae family and is caused by its various human pathogenic species (L. danovani, L. infantum, L. chagasi, L. tropica, L. major, L. braziliensis, L. mexicana) visceral, cutaneous or the mucosal leishmaniasis. L. affects only the endolysosomal compartments of macrophages. The immune response is based solely on CD4 ⁺ cells.

Heinzel et al. (Heinzel, FP, Sadick, MD, Holaday, BJ Coffman, RL and Locksley, RM; J. Exp. Medecine 169, 59-72, 1989) and Kemp et al. (Kemp, M., Theander, TG and Kharazmi, A; Immunology Today 17, 13-16, 1996) used the experimental mouse model, which reflects the human immunological conditions, to have two different resistant breeding lines (C57BL / 6 or C3H / HeN) infected with L. major. It is known from the document cited above that the TH1 subclass of the CD4 ⁺ cells is responsible in particular for the resistance of the two breeding strains.

From the writings of Heinzel, F.P., Sadick, M.D., Holaday, B.J., Coffman, R.L. and Locksley, R.M., Journal of Experimental Medicine 169, 59-72, 1989, and Sadick, M.D., Locksley, R.M., Tubbs, C. and Raff, H.V .; J. Immunot. 136, 655- 661, 1986; is also known to be genetically non-resistant Mouse strains such as Balb / c immunologically for Leishmania infection react by activating the TH2 cells, which, however, act as a signal generator for the Antibody production that Leishmania infection cannot adequately fight. Kemp, M., Theander, T.G. and Kharazmi, A. (Immunology Today 17, 13-16, 1996) have shown that such with human infection with L. - according to the Mouse model - one that corresponds to resistance or non-resistance Activation pattern of TH1 or TH2 cells exists.

From the writings of Mosmann & Coffman (Mosmann T. R. and Coffman, R. L., Annu. Rev. Immunol. 7, 145, 1989) is known to be a characteristic pattern accompanied by activation of TH1 or TH2 cells by cytokines. Something like that TH1 cells in particular produce interferon-γ (IFN-γ), while TH2 cells Produce interleukin-4 (IL-4).

It was used for both the mouse model (Kemp, M., Theander, T.G. and Kharazmi, A; Immunology Today 17, 13-16, 1996) as well as by clinical experiments shown (Gaafar, A., Kharazmi, A., Ismail, A., Kemp, M., Hey, A., Christensen,  C. B., Dafalla; M., el Kadaro, A.Y., el Hassan and Theander, T.G .; Clin. Exp. Immumology 100, 239-245, 1995) that the interferon-γ production the Severity of Leishmania infection determined.

From the content of the Badaro & Johnson script (Badaro, R and Johnson, W. D., J. Infect. Dis. 167, S13-S15, 1994) it can be seen that clinical trials, adequate treatment of a human by the administration of interferon-γ Providing leishmania infection did not have the expected success showed. The functional immune defense could not by this cytokine administration be restored.

Among other infections, itracellular parasitic pathogens which can trigger TH1 / TH2 diseases in the sense of the invention are to be understood in particular as follows:
Trypanosomoses (Trypanosoma cruzi, Trypanosoma equiperdium), Toxoplasmoses (Toxoplasma gondii), Mycosis (Mycosis fungoides), Candida infections (Candida), Tubercolosis (Mycobacterium tuberculosis), Lepra Mycobacterium leprae, petrenis (Listeria), Bordetella (Bordetella (Chlamydia) and BCG (bacille Calmette-Guérin).

In the publication WO95 / 13293 A1, peptides from FasL are described which are linked to the Tie FasR. These peptides are used in the treatment of viral and Autoimmune diseases used.

It is also known from the literature that interferon-γ can upregulate the expression of FasR on CD34 ⁺ cells of the bone marrow (Maciejewski, J .; Seller, C .; Sato, T .; Anderson, S .; Young; N .; British J. of Haemotology, 1995, 91, 245-252). This relationship has been demonstrated for the clinical picture of aplastic anemia.

The object of the present invention is to use more suitable ones Provide adequate medicinal products or new compositions of substances that modulate the immune defense in TH1 / TH2 diseases such that intracellularly parasitizing bacterial or protozoological pathogens by an effective, of the immune response carried by the inflammatory TH1 cells.

The present invention relates to the use of FasL Claim 1.

There is a reduced TH1 response in TH1 / TH2 diseases. For the Immunological defense is particularly important for infections with intracellular A crucial factor is an effective inflammatory TH1 cell response. According to the invention, the administration of FasL results in the immune response of the TH1 cells compensated for diseases such as leishmaniasis, listeriosis, Trypanosomosis, Bortedella, Leptra etc. the intracellularly residing pathogens can effectively combat bacterial or protozoological origin. The The addition of FasL thus compensates for a missing or insufficient TH1 Immune response. This makes the use of FasL for use as a drug in TH1 / TH2 diseases.

It is used as a therapeutic agent for the treatment of TH1 / TH2 diseases the gene product of the human FasL gene sequence. Also physiologically active and Apoptosis potent gene products from FasL gene segments, e.g. B. N- or C-terminal shortened gene products, or gene products that have one or more intracistronic Gene segments are missing and are suitable for application.  

The use of FasL for the treatment of TH1 / TH2 diseases can be found in the Veterinary medicine in mammals, especially in farm animals, especially in Pork, beef and sheep, but also in pets, especially in dogs and cats, and in human pathology to support the TH1 immune response intracellular bacterial or protozoological pathogens occur. Among farm animals are to be understood in particular those mammals that humans approach Transport tasks or in the agricultural sector for food extraction served.

The use of FasL for the treatment of human is particularly preferred Leishmaniasis, being both cutaneous and visceral as well as mucous membrane leishmaniases caused by L. donovani, L. infantum, L. chagasi, L. tropica, L. major, L. braziliensis, L. mexicana are triggered. Corresponding In livestock, the intracellularly parasitic Leishmania species with FasL Dosages are combated.

FasL initiates apoptosis in infected cells. One embodiment represents the Use of FasL in pharmacological form in vivo. In vitro can in a another embodiment, the use of FasL in the course of an external blood wash respectively. Infected blood cells, i.e. differentiated cells of the hematopoietic Systems, especially macrophages, are killed ex vivo by administration of FasL. The blood treated with FasL is again fed to the patient. FasL will So in this case applied externally to avoid any undesirable side effects to avoid giving FasL.

In a preferred embodiment, FasL is applied topically to the patient administered. With topical application is the non-systemic application meant. The substance according to the invention is then applied to the epidermis or instilled in the eyes, nose, or ears, but essentially not gets into the bloodstream.  

Liquid, semi-liquid or semi-solid formulations are suitable for topical application. They will cut in on the cutaneous or mucosal lesions applied in a resorbable form. For example, there are creamy ones Formulations, ointments, pastes, drops or lotions are available. Also in solid form, the topical application of FasL takes place through the provision of Powder.

Ointments, pastes or cream formulations contain the active substance as aqueous or non-aqueous solution or suspension based on a fatty or non-fatty carrier.

In another preferred embodiment, FasL is administered systemically, with oral, intravenous, intraperitoneal and intramuscular Offer forms of administration. The formulation for one is particularly preferred parenteral application.

The carrier materials used for systemic application are in particular the the following dosage forms (Hartke & Mutschler, German Pharmacopoeia, 9th Edition, 1986, Volume 3 "Parenteralia", pp. 2670-2677, Scientific Publishing Company Stuttgart, Govi-Verlag GmbH Frankfurt; Pharmacopoea Helvetica, Edition Septima 1.1.1991 and renewed version 1996, Monograph "Parenteralia"; U.S. Pharmacopeia, National Formulary 18, 1995, S. 1650-1652).

FasL is given to the patient depending on the type of application (especially depending on systemic or topical application, application in vivo or ex vivo), on the adjuvants, the respective galenical preparation, the type of infection, the stage of infection and the current course of infection as well as on the known clinical parameters , for example on the patient's age and the specific patient's medical history, in a dose between 1 picogram and 100 mg per kg of body weight (of the patient). Application in a concentration between 10 ^-8 and 10 ^-3 g / kg body weight is preferred.

In a particularly preferred embodiment, the active FasL substance is included combined other pharmacological carrier materials, for example the Improve pharmacokinetic properties, but at the same time physiologically are tolerated.

Human FasL ^- / CD4 ⁺ / TH1 cells or FasL only weakly expressing cells can be transfected with FasL according to the following methods. The introduction of FasL gene material can take place by lipofection, microinjection, with the help of viral vectors or by electroporation. The FasL gene material can be provided with an upstream promoter and can be transfected in this form or can be inserted in a targeted manner at the position of the physiological promoter. Clonogenic cells that are transfected by standard methods and stably express FasL are used therapeutically for TH1 / TH2 diseases in order to strengthen the TH1 immune response.

For this purpose, T-lymphocytes are taken from the infected patient and such TH1 cells isolated, whose T cell receptors antigens of the respective infection recognize pathogen. These cells are cultured and with FasL gene material transfected. The FasL gene material can be both the human FasL gene sequence include as well physiologically active and potent for apoptosis reaction FasL gene segments or gene segments, the N- or C-terminal shortened FasL protein express. Stable transfected, clonogenic, autologous cells become the patient with FasL deficiency or a suppressed TH1 immune response applied. In this particular embodiment, transfected with FasL serve TH1 cells for the treatment of TH1 / TH2 diseases. The same thing Infection spectrum covered, already mentioned above for the treatment it FasL has been. The transfected, autologous cells mobilize the FasL / FasR  Apoptosis system and kill infected cells, especially macrophages.

It has been found according to the invention that TH1 lymphocytes with Leishmania can kill infected macrophages in vitro.

To test this potency of TH1 lymphocytes experimentally, Leishmania-infected and activated macrophages were cocultivated with Leishmania-induced TH1 cells in the presence of interferon-γ. Lymph nodes from C57BL / 6 mice that had previously been infected with Leishmania major for 5 to 8 weeks were removed from the TH1 cells by removing the lymph nodes that dispose of the lesions and removing the TH1 cells by the MACS- ^* (Magnetic Cell Sorter) processes (Miltenyi Biotech GmbH, Bergisch-Gladbach). This material was obtained in vitro by L. major promastigotes (previously treated with UV radiation) in the presence of an irradiated (3000 rad) normal lymph node population in which the CD4 ⁺ / T cells were eliminated and which presented as a source of antigen Cells (APC) served, restimulated. The TH1 cell type was confirmed by measuring interferon-γ and interleukin 2- (IL-2) activity. After 72 hours, the CD4 ⁺ "blast" cells ("blast" cells are cells that have undergone clonal expansion after stimulation) were isolated and washed. They were co-cultured with bone marrow macrophages that had previously been infected with L. major in a ratio of 1: 1 and in the presence of interferon-γ (833.5 nkat / ml). It was found that after 6 hours of coculturing, 60% of the macrophages from the C57BL / 6 strain were apoptotic. On the other hand, those macrophages that were treated analogously, but were taken from the syngenic, FasR-deficient mouse breeding strain lpr, showed almost no apoptotic features.

In order to check the function of the FasL / FasR-controlled apoptosis signal in more detail, tests were also carried out according to the invention in vivo with the mouse breeding strains C57BL / 6 (resistant strain) and the syngeneic mouse strains gld (FasL ^- ) and lpr (FasR ^- ), all with Leishmania major promastigotes (in the stationary phase) had been subcutaneously infected in a dose of 2 × 10 ⁶ (in a final volume of 50 μl).

For all three strains mentioned, the size of the lesions on the The paws of the mice develop the Leishmania infection once a week tracked and measured with a caliper by the size of the infected paw was compared with the size of the opposite, non-infected paw.

The present invention further relates to the use according to claims 7 and 8. The use of a corresponding invention Claimed composition of substances to combat TH1 / TH2 diseases, for example for use in the treatment of leishmaniasis, listeriosis or infections with other intracellular bacterial or protozoological Pathogens or in a preferred form for the treatment of cutaneous, visceral or mucosal leishmaniasis.

The invention also relates to the use of autologous CD4 ⁺ / FasL ^- / TH1 cell lines of the patient transfected with the FasL gene sequence or physiologically active sections of the FasL gene sequence for combating TH1 / TH2 diseases, for example the use of with FasL transfected CD4 ⁺ / FasL ^- / TH1 cell lines for combating leishmaniasis, listeriosis or infections with other intracellular bacterial or protozoological pathogens, in particular the use of CD4 ⁺ / FasL ^- / TH1 cell lines transfected with FasL for combating cutaneous, visceral or Phlegm is claimed to be skin-leukemia.

The invention is explained in more detail by the following figures.  

In Fig. 1 it is shown that the resistant control strain C57BL / 6, as expected, showed almost no signs of the lesions 12 weeks after the infection event. In contrast, it is also clear from FIG. 1 that a progressive course of the size of the lesions can be observed in the two other mouse strains lpr and gld. This experiment underlines the importance of functional FasL and FasR for an effective inflammatory TH1 effector function. The axes represent the number of weeks after the infection event on the abscissa and the lesion size in mm on the ordinate.

In Fig. 2 it is shown by series of dilutions that the number of Leishmania parasites in the lesions of the lpr or gld mice is significantly increased compared to samples from the lesions of the mouse control group which have a functional FasL / FasR signal transfer system (C57BL / 6) . The number of parasites per mg tissue is plotted on the abscissa. The number of parasites was determined after 30 and 94 days, respectively, for each of the three mouse strains (exception: lpr mice). For the lpr mice, the number of parasites was not determined after 90 days (ND).

In Fig. 3 it is shown that after an in vitro restimulation of the TH1 cells in all three mouse strains (control group, lpr and gld) a comparable production of interferon-γ can be demonstrated.

In contrast, the IL-4 characteristic of TH2 cells (not in the figures shown) almost undetectable. This proves experimentally that both Mutant strains with regard to cell proliferation and cytokine production Have wild type properties. In addition, this experiment shows that at the mutant strains an adequate cytokine production for the initiation of a TH1 immune response is present.

Fig. 4 shows that potential influences of the immunologically effective CD8 ⁺ T cells with their characteristic cytotoxic effector function by Perforin- secretion at a Leishmania infection may be excluded. A perforin-deficient mouse strain shows the same kinetic profile in eliminating the infectious agent compared to wild-type strains. The axes represent the number of weeks after the infection event on the abscissa and the lesion size in mm on the ordinate.

In FIG. 5, the size of the lesions in function of the time after the original Leishmania infection with L. major at gld mice, known FasL-deficient (1) after administration of FasL in the form of a supernatant of FasL-expressing cell cultures (2) without FasL and (3) in control C57BL / 6 mice who also did not receive any FasL injections. The number of weeks after infection is plotted on the abscissa, and the lesion size in mm is plotted on the ordinate. For this purpose, gld mice were infected with L. major (5 weeks of infection) and then in the case of (1) in the following three weeks with 50 µl of a 5-fold solution of the supernatant from FasL-producing neuro-2a cell cultures treated for four days. The FasL-containing solution was injected into the lesions in case (1).

Those gld mice that were not injected with FasL show nine weeks after Infection an average lesion size of 1.3 mm. Have against it Wild-type mice with functional FasL and FasR production even without administration of FasL only an average lesion size of 0.3 mm after the same time. Mice of the gld strain who received injections with FasL samples show a significantly reduced lesion size compared to the untreated gld mouse of an average of 0.75 mm

In FIG. 6, the number of parasites per mg of tissue, and is the abscissa for the gld mouse strains treated with FasL or with a control sample, plotted for the control strain C57BL / 6. The result of this experiment is comparable to the result from FIG. 5. Seven days after the last injection of FasL samples or control samples from supernatants from sham-transfected neuro-2a cells into the gld mice, more than 40 parasites in 1 mg tissue were determined for the mice treated with Fasl control samples. Only about eight parasites / mg tissue were detected in the gL mice treated with FasL. The control group of resistant C57BL / 6 mice had almost no parasites in their lesions 63 days after the infection event.

The results of these experiments, shown in FIGS. 5 and 6, can be summarized in such a way that only an interaction of functional FasR with functional FasL genes or their functional expression products is an effective immunological defense against the Leishmania infection on the basis of the Ensures TH1 reaction.

Is known from the literature (Swihart, K., Fruth, U., Messmer, N., Hug, K., Behin, R., Huang, S., Del Giudice, G., Aguet, M. and Louis, J.A .; J. Exp. Med. 181, 1995) that interferon-γ (IFN-γ) contribute to the effectiveness of the immune response Leishmaniases are of crucial importance. Mouse strains with genetically resistant disposition to leishmaniasis shows an infauste Course of infection as soon as they do not have a functional IFN-γ receptor. In various documents (Green, S.J., Nacy, C.A. and Meltzer, M. S., J. Leukoc. Biol. 50, 93-103, 1991; Mauel, J., Betz-Corradin, S, and Buchmüller- Rouiller, Y .; Res. Immunol. 7, 145, 1989; Wei, X.-Q., Charles, I.G., Smith, A., Ure, J., Feng, G.-J., Huang, F.-P., Damo, X., Muller, W., Moncada, S. and Liew, F. Y .; Nature 375, 408-411, 1995) the effect of IFN-γ on induction of NO synthase recycled, which increases NO synthesis.

It has been found that IFN - γ is the expression of FasR on macrophages which are associated with Leishmania are infected.

FIG. 7 shows that the expression of FasR is increased both in the wild type and in gld mice after administration of IFN-γ. In contrast, this effect is not detectable in the FasR-deficient lrp mice.

Bone marrow macrophages were six week old mice of the strains  Bone marrow macrophages were six week old mice of the strains C57BL / 6, lpr and gld taken and according to the information in Feng et al. (Feng, Z. Y., Louis, J., Kindler, V., Pedrazzini, T., Eliason J.F., Behin, R. and Vasalli, P .; Eur. J. Immunol. 18, 1245-1251, 1988). After that, the Macrophages with promastigotes from Leishmania major in a ratio of 5: 1 infected, washed after 12 h and more in the presence of IFN-γ (833.5 nkat / ml) Raised 48 hours in culture. After labeling with anti-Mac1 monoclonal Antibodies M1 / 70 showed a positive signal in 98% of the macrophages. The Macrophages were also treated with a control antibody, a biotinylated one Anti-Fas antibody (JO2, Pharmingen, USA) or a biotinylated monoclonal anti-MHC-II antibody (monoclonal Ak BP107) labeled and then added with fluorescent streptavidin (Caltag, USA). The analysis was measured on a FACScanII flow cytometer (Becton Dickinson, Mountain View, CA, USA).

It was also demonstrated that the activation of the Expression of FasR on the macrophages to an increased sensitivity of the cells leads to an apoptosis controlled by FasL. It became experimental detection activated with IFN-γ and infected with Leishmania Macrophages from the three strains of mice mentioned above were used. These cells were reared for 48 hours in culture as described in the last paragraph and then with the supernatant from recombinant FasL-producing neuro- 2a cells or from sham-transfected neuro-2a cells (Rensing-Ehl, A., Frei, K., Flury, R., Matiba, B., Mariani, S. M., Weller, M., Aebischer, P., Krammer, P. H. and Fontana, A .; Eur. J. Immunol. 25, 2253-2258, 1995). Apoptotic Cells were extracted using subdiploid DNA using the method of Renno et al. (Renno, T., Hahne, M., Tschopp, J. and MacDonald, H.R., J. Exp. Med. 183, 431-437, 1996).  

TH1 cells can use the FasL / FasR system to induce apoptosis Macrophages with Leishmania or other intracellular infectious agents are infected, if and insofar as the macrophages are previously infected with IFN-γ have been activated. IFN-γ production to stimulate NO synthesis is not sufficient according to the present invention to carry out the experiments the immune responses of the mouse strains C57BL / 6, gld and lpr to the Explain pathogen Leishmania.

In Fig. 8 it is shown that the macrophages of the three strains have an approximately comparable NO synthesis rate after the addition of IFN-γ. Thus, according to the invention, in addition to the known mechanism of NO production, FasR expression is also activated as a result of appropriate stimulation by IFN-γ to combat intracellular infection in antigen-presenting cells.

The present invention is explained in more detail by means of exemplary embodiments.

First of all, the following exemplary embodiments are the basis experimental boundary conditions explained:

The mouse strains C57BL / 6 and C3H / HeN were purchased from IFFA-CREDO (Saimt Germain-sur-l'Abresle, France). The two deficient mouse strains gld (FasL ^- ) and lpr (FaaR ^- ) are derived from the strains C57BL / 6 or C3H / HeN and were bred in Jackson's laboratory (Bar Harbor, Maine, USA). All mice were six to eight weeks old at the time of the experiments.

The Leishmania parasites of the L. major strain LV 39 (MRHO / SU / 59 / P strain) were preserved in vivo and in vitro using the method of Louis, J.A., Moedder, E., Behin, R., Engers, H.D. (Eur. J. Immunol., 9, 841-847, 1979).  

The infection of the mice with the parasites took place subcutaneously in a paw with 2 × 10 ⁶ promastigotes in the stationary phase with a final volume of 50 μl.

The recombinant FasL was in neuro-2a cells by the method of Rensing- Ehl, A., Frei, K., Flury, R., Matiba, B., Mariani, S. M., Weller, M., Aebischer, P., Krammer, P.H. and Fontana, A. (Eur. J. Immunol. 25, 2253-2258, 1995).

The apoptotic cells were analyzed using the method of Renno, T., Hahne, M., Tschopp, J. and MacDonald, H.R. (J. Exp. Med. 183, 431-437, 1996) by the Content of subdiploid DNA determined.

The bone marrow macrophages come from bone marrow progenitor cells (after in vitro differentiation) from the method of Feng et al. (Feng, Z. Y., Louis, J., Kindler, V., Pedrazzini, T., Eliason, J.F., Behin, R., and Vasalli, P .; Eur. J. Immunol. 18, 1245-1251, 1988).

Cultivation of the lymphocytes: The cells were taken from those lymph nodes associated with the L. major-related lesions (3 × 10 ⁶ / ml) and in vitro with UV-irradiated L. major promastigotes (1 × 10 ⁶ / ml) stimulated for 72 h or with ConA (2.5 mg / ml) for 48 h on the bottom of microtiter plates. 37 ° C, an air environment enriched with 7% CO ₂ and a final volume of 200 µl were chosen as external conditions. The cells were in DMEM (Dulbecco's Modified Eagles Medium) with 5% heat-inactivated FCS (Fetal Calf Serum), with L-glutamine (216 mg / ml), 5 × 10 ^-5 mol / l β-ME, 10 mmol / l Hepes, 1,667.10 ^-6 cat / ml penicillin and 100 mg / ml streptomycin. The supernatants from appropriate cultures were pooled and stored at -20 ° C until they were used for cytokine determination. The CD4 ⁺ T cells were removed for certain experiments in the cell suspensions by adding monoclonal Ig-M antibodies against the CD4 marker (RL 172.4) and low-tox rabbit complement (Cedarlane, Horny, Ontario , Canada) using the method of Swihart, K., Fruth, U., Messmer, N., Hug, K., Behin, R., Huang, S., Del Giudice, G., Aguet, M. and Louis, JA (3rd Exp. Med. 181, 1995) were used. In some experiments, the level of proliferation was also checked by measuring the incorporation of ³ H-methylthymidine ( ³ htdr, Amersham, GB).

a)

1. Experimental execution:

An in vitro test system was used to demonstrate the apoptotic effect of CD4 ⁺ / TH1 cells on bone marrow macrophages infected with L. major.

The CD4 ⁺ / TH1 cells were taken from mice of the breeding strain C57BL / 6 (IFFA-CREDO, Saint Germain-sur-l'Abresle, France), which had been infected with L. major 5 to 8 weeks previously.

The lymph nodes in the area of the Leishmania lesions were removed and cleaned up using the MACS method according to the method specified by the manufacturer (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany). The purified CD4 ⁺ cells (2 × 10 ⁵ per well) were stimulated in vitro by L. major promastigotes (previously treated with UV radiation) in the presence of irradiated (3000 rad) lymph node cell populations without CD4 ⁺ cells to use them as material for antigen presenting cells (APC). After 72 h, the CD4 ⁺ "blast" cells (see above) were isolated and washed. They were co-cultured with bone marrow macrophages that had previously been infected with L. major and activated with interferon-γ according to the method described above in a ratio of 1: 1 (in a final volume of 2 ml in Petri dishes). After 6 h of coculturing, the bone marrow macrophages were harvested with a plastic scraper, washed and the proportion of apoptotic cells was determined by FACS analysis. For this purpose, the macrophage population was labeled with monoclonal anti-Mac1 antibodies M1 / 70. After labeling, 98% of the macrophages showed a positive signal. The macrophages were also labeled with a control antibody, a biotinylated anti-Fas antibody (JO2, Pharmingen, USA) or a biotinylated monoclonal anti-MHC-II antibody (BP107) and then fluorescent streptavidin (Caltag, USA) was added . The analysis was carried out on a FACScanII * (Fluorescence Analysis Cell Sorter) flow cytometer.

2. Result

It was found that after 6 hours of cocultivation, more than 60% of the Macrophages from the C57BL / 6 strain were apoptotic. Against that pointed Macrophages treated analogously, but the syngeneic, FasR-deficient Mouse breeding strain lpr were taken, almost no apoptotic Characteristics on (approx. 2% of the cells).

b)

1. Experimental execution

Evidence that TH1 / TH2 disease is treated effectively with FasL has been demonstrated in various strains of mice infected with L. major. For this purpose, gld mice were infected with L. major 5 weeks earlier were, with a 5-fold concentration every four days over the course of three weeks Supernatant from FasL-expressing neuro-2a cells treated. In addition, 50 µI injected into the Leishmania lesions. The lesion size was increased during the entire experiment over nine weeks. The size of the lesions was made using a caliper gauge, which was a measurement of the thickness of the infected in the Compared to the thickness of the opposite, non-infected paw allowed, determined. Control mice that were also infected were challenged with a Supernatant treated by neuro-2a sham transfectants that do not have FasL express (Rensing-Ehl, A., Frei, K., Flury, R., Matiba, B., Mariani, S. M., Weller, M., Aebischer, P., Krammer, P.H. and Fontana, A .; Eur. J. Immunol. 25, 2253-2258, 1995). Seven days after the end of treatment (or nine Weeks after infection) the lesion size and the number of parasites were in  the lesions in the gld mice treated in this way. The number of Parasites in the lesions on the infected paws were identified by one Dilution test at specific intervals according to the method of Titus, R. G., Marchand, M., Boon, T. and Louis, JA. (Parasite Immunology 7, 545-555, 1985) certainly.

2. Result

The result of the treatment of FasL-deficient gld mice with FasL is plotted in FIGS . 5 and b. Figure 5 shows the lesion size as a function of time (in weeks after infection). The three curves symbolize 1. (∎) gld mice that were treated with FasL from FasL-expressing cells five weeks after infection, 2. () gld mice that were treated with the supernatant from FasL mock transfectants and 3. ) (o) Control mice from strain C57BL / 6.

Five weeks after infection and before treatment with the various The gld mice show a lesion size of approx. 0.9 mm. While in further observation period the lesion size in the gld mice, which none functional FasL was injected, increasing (to approximately 1.2 mm nine weeks after the Infection), the lesion size decreases in the mice with functional FasL were treated. These mice only show nine weeks after infection an average lesion size of 0.6 mm.

This experiment documents that the treatment of FasL deficient gld mice have a clear therapeutic effect on the Infection course.

FIG. 6 shows the number of parasites per mg tissue determined in the lesions of the C57BL6 mice, the gLl mice treated with FasL and the gLD mice treated with the supernatant of transfectants, as determined by a dilution assay. The number of parasites was determined nine weeks after infection and seven days after the end of treatment.

The number of parasites is significantly reduced by the injection of FasL (less than 10 parasites per mg tissue). In contrast, they do not use FasL treated mice with more than 40 parasites per mg tissue. So that too the experimental effect of FasL in this Treatment of TH1 / TH2 diseases, here using the example of Leishmania infection, clear.

c)

1. Experimental execution

The dependence of the mechanism of apoptosis on intracellularly infected cells to describe the presence of interferon-γ and thus the interferon-γ- To document activity for the Fas mechanism, L. major infected bone marrow macrophages treated with IFN-γ and finally on the Expression of appropriate marker proteins examined.

For this purpose, bone marrow cells from C57BL / 6, gld and lpr mice, the gld and lpr mice having a C57BL / 6 origin, were placed in 10 ml plastic petri dishes in DMEM (see above) with the addition of 20% horse serum and 30% serum-free supernatant from L929 cells, as donors of M-CSF. After seven days of cultivation, the macrophages were detached and suspended in DMEM with 10% FCS (see above), L-glutamine (216 mg / ml), 10 mmol / l Hepes, 1,667.10 ^-6 cat / ml penicillin and 100 mg / ml streptomycin. The macrophages were then infected with L. major promastigotes in a ratio of 5: 1 and taken up in a final volume of 2 ml in plastic petri dishes. The bone marrow macrophages were washed twelve hours after infection and incubated in the medium described above for 48 h with recombinant IFN-γ (833.5 nkat / ml). During the last four hours of incubation, the supernatant from either FasL-producing neuro-2a cells or neuro-2a sham transfectants (20% by volume) was added to the macrophage cultures. In the case of macrophages, the cell surface expression of MHC-II (main histocompatibility complex of class II) and FasR molecules were finally determined by the FACS method described above (see under a).

2. Result

FIG. 7 shows the result of this experiment as a plot of the cell number against the number of Co (control), MHC-II and FasR molecules for the wild-type, the gld and the gld mice.

The comparable activation of the three mouse strains by IFN-y was shown by the proven expression of MHC-II molecules. With the three tribes is the integral of the cell number and the number of expressed MHC-II molecules comparable. The number of FasR molecules expressed as a result of the treatment with IFN-γ, however, affects the individual strains with characteristic Differ from. Both the wild type strain and the gld strain show one increased expression rate, but not the FasR-deficient gld strain.

It is thus clear that treatment with IFN-γ has a direct influence on the FasL / FasR-controlled apoptosis signal has, by the expression rate of the FasR- Molecules are upregulated by IFN-γ.

Claims (11)

1. Use of FasL or one that is potent for apoptosis FasL segment as an active pharmacological inventory part to combat TH1 / TH2 diseases, out triggers through pathogens residing intracellularly taken viral pathogens. 2. Use of FasL or one that is potent for apoptosis FasL segments according to claim 1 for combating Leishmaniasis, listeriosis, tuberculosis or infection with other intracellular bacterial or protozoological pathogens. 3. Use of FasL or one that is potent for apoptosis FasL segments according to claim 1 or 2 for combating from cutaneous, visceral or mucosal leishmanio sen. 4. Use of FasL or one that is potent for apoptosis FasL segment according to one of claims 1 to 3 Apoptosis induction ex vivo. 5. Use of FasL or one that is potent for apoptosis FasL segments according to one of the above claims che in homogeneous form with a physiologically ver inert pharmaceutical carrier material in topi shear or systemic application. 6. Use of FasL or a FasL segment potent for apoptosis according to one of the above claims in a concentration range between 10 ^-12 and 10 ^-1 g / kg body weight, preferably between 10 ^-8 and 10 ^-3 g / kg body weight. 7. Use of FasL or one that is potent for apoptosis FasL segment according to one of the preceding claims in Combination with one or more cytokine (s), especially interferon-γ. 8. Use of a composition of matter according to claim 7 for in vivo treatment. 9. Use of autologous CD4 ⁺ / FasL ^- / TH1 cell lines of the patient transfected with the FasL gene sequence or physiologically active sections of the FasL gene sequence for combating TH1 / TH2 diseases triggered by intracellularly residing pathogens viral pathogens. 10. Use according to claim 9 for combating Leishmaniasis, listeriosis, tuberculosis or infection with other intracellular bacterial or protozoological pathogens. 11. Use according to claim 10 for combating cutaneous, visceral, or mucosal leishmaniasis.