Note: Descriptions are shown in the official language in which they were submitted.
CA 02269034 1999-04-16
'.
1
Application of Fast or CD4+/FasL-/TI31 cell lines transfected with Fast
for the treatment of THl/TH2 diseases. Method for the preparation of
CD4+/FasL+/THl cell lines and material composition of Fast and one or
several cytokines as well as their application for the treatment of
TH1/TH2 diseases.
The invention concerns the application of Fast for the treatment of
TH1/TH2 diseases, especially leishmaniosis, AmS, listeriosis, a method for
the preparation of CD4+/FasL+/THl cell lines from CD4+/Fasl~/TH1 cell
lines, their application for the treatment of TH1/TH2 diseases, a material
composition containing Fast and one or several cytokines, as well as their
2 0 application as drugs, especially for the treatment of TH1/TH2 diseases.
TH1/TH2 diseases include a spectrum of immunologically caused disorders,
which are based on a shift of the immune response or of an inadequate
immune response after infection by pathogens, for example, viruses, bacteria
2 5 or protozoa.
The immune system of animals or humans responds to infection with
pathogens by a cellular and/or a humoral immune reaction, depending on
specific characteristics of the pathogen. While the cellular immune response
CA 02269034 1999-04-16
2
is used mostly against intracellular parasites, the humoral immune response
is characterized by the production of antibodies, which neutralize the
pathogens extracellularly. B-lymphocytes participate especially in the
humoral immune response, while the class T-lymphocytes participate both in
cellular as well as humoral immune response. Therefore, one distinguishes
between two different subclasses of T-lymphocytes, namely CDS+ cells,
which carry a surface molecule designated with CDB, and analogously a
subclass (CD4+ cells), which have the CD4 marker. These T-cells which
carry CD4 are also called T-helper cells (TIT). _
T-helper cells are again subdivided into two subclasses, into the so-called
TH1 cells and TH2 cells. Both cell classes occupy central positions in the
combatting of intracellular or extracellular pathogens. The inflammatory
TH1 cells serve as immune response against intracellular pathogens, such as
Leishmania, viruses or intracellular parasitary bacteria, such as Listeria. On
the other hand, Thi2 cells cause activation of B-lymphocytes, which - as
mentioned above - control the humoral immunity against extracellular
pathogens.
2 0 For adequate immune response to infection, it is of decisive importance
that
the TH1- or TH2-guided immune response predominates, depending on the
type of pathogen. If, in an infection with intracellular parasitary pathogens,
the THZ-guided immune response dominates, that is, if sufficient Tlil-
guided immune response is lacking or is deficient, then in the sense of this
invention, we are dealing with a TH1/TH2 disease.
It is known from the literature that TH1 cells exert a cytolytic action
through
a pathway controlled by Fas ligands (Fast) or Fas receptor (FasR) (Ashany,
D., Song, X., Lacy, E., Nikoliczugic, J., Friedman, S. M. and Elkon, K.
CA 02269034 1999-04-16
3
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). Moreover,
it was described that cloned TH1 cells have a high expression rate of Fast,
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). When Fast is bonded to its
receptor, FasR, a cascade of signals is triggered, which finally lead to cell
death (apoptosis) (Nagata, S. and Golstein; P., Science 267, 1449-1456,
1995). With the triggering of the death of an infected cell, the
multiplication cycle of the intracellular pathogen is also interrupted.
Furthermore, it is described that, as a result of the activity of the
inflammatory TH1 cells, to give off Fast in a direct manner, various target
cells of greatly varied tissues, including macrophages, can be killed in vitro
2 0 as a result of the expression of FasR (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 the class of pathogens which cause
intracellular infection.
CA 02269034 1999-04-16
4
Leishmania (L.) belongs to the family of Trypanosomatidae and causes
visceral, cutaneous or mucosal leishmanioses through its various human-
pathogenic species (L. donovani, L. infantum, L. chagasi, L. tropica, L.
major, L. braziliensis, L. mexicana). L. attacks exclusively the
endolysosomal compartments of macrophages. The immune response is
based only on CD4+ cells.
Heinzel et al. (Heinzel, F. P., Sadick, M. D., Holaday, B. J., Coffman, R.
L. and Locksley, R. M.; J. Exp. Medecine 169, 59-72, 1989) and Kemp et
al. (Kemp, M., Theander, T. G. and Kharazmi, A.; Immunology today 17,
13-16, 1996) have infected two different resistant bred strains (C57BL/6 or
C3H/Hel~ with L. major using the experimental mouse model for
leishmanioses that reflects the human-immunological relationships. It is
known from the document cited above, that especially the TH1 subclass of
the CD4+ cells is responsible for the resistance of both bred strains.
Moreover, it is known from the papers 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 Ruff, H. V.; J. Immunol. 136, 655-661, 1986; that the
genetically nonresistant mouse strains such as Balb/c, react immunologically
to the Leishmania infection with activation of the TH2 cells, but these cannot
combat the Leishmania infection as signallers for antibody production.
Kemp, M. , Theander, T. G. and Kharazmi, A. (Immunology today 17, 13-
2 5 16, 1996) showed that in human infection with L. - corresponding to the
mouse model - an activation pattern of TH1 or TH2 cells exists which
correspond to resistance or nonresistance.
._ ____.__ .. _._._ _ . ._ . .. . _. .._.._ _ . .. _.__. .. . . __ _ _ . ._ .
_ .. _ o .___ .. __.. _ __ ._ .. ___ . _
CA 02269034 1999- 4 16
From the publication of Mosmann & Coffman (Mosmann, T. R. and
Coffman, R. L., Annu. Rev. Immunol. 7, 145, 1989) it is known that a
characteristic pattern of cytokines accompanies the activation of TH1- or
TH2 cells. Thus, TH1 cells produce especially y-interferon (IFN-y), while
5 TH2 cells produce interleukin-4 (ZL-4).
It was shown both for the mouse model (Kemp, M. , Theander, T. G. and
Kharazmi, A.; Immunology today 17, 13-16, 1996) as well as by clinical
experiments (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.; Clip. Exp. Immunology 100, 239-245, 1995), that the production of
y-interferon determines the degree of severity of the Leishmania infection.
From the content of the paper of Badaro & Johnson (Badaro, R. and
Johnson, W. D., J. Infect. Dis. 167, S13-515, 1994) one can see that
clinical attempts, to make adequate treatment of human Leishmania infection
available by the administration of y-interferon, did not show the expected
success. The functional immune defense could not be restored by
administration of this cytokine.
Among others, infections by intracellular parasitary pathogens, which can
..
trigger TH1/TH2 diseases according to the invention, include especially the
following: trypanosomoses (Trypanosome cruzi, Trypanosome equiperdium),
toxoplasmoses (Toxoplasma gondii~, mycosis (Mycosis fungoides), Candida
infections (Candida), tuberculosis (Mycobacterium tuberculosis), leprosy
(Mycobacterium leprae), bordetella (Bordetella pertussis), listerioses
(Listeria), chlamydioses (Chlamydia) and BCG (Calmette-Guerin bacillus).
CA 02269034 1999-04-16
6
In the documents of Clerici and Shearer (Clerici, M. and Shearer, G. M.;
Immunology Today 14, 107-111, 1993) and Mosmann (Mosmann, T. R.;
Science 265, 193-194, 1994) it is described that immunologically
problematic situations arise when, as a result of HIV infection, CD4+/TH2
cells dominate the immune response. Only TH1 cells permit an effective
elimination of the infected cells of the immune system or of intracellularly
occurring viruses. Thus, the immune weakness that occurs during AIDS
disease is a TH1/TH2 disease in the sense of the present invention.
The task of the present invention is to provide an adequate drug by using
suitable materials or by the introduction of new material compositions,
which modulate the immune response in TH1/TH2 diseases in such a way
that intracellular pathogens are attacked by an effective immune response
carried by the inflammatory TH1 cells.
Within the framework of the present invention, material compositions and
the use of these compositions or other substances for the treatment of
TH1/TH2 diseases are disclosed.
2 o Therefore, the object of the present invention is the use of Fast as an
active
pharmacological component for the treatment of ThI1/TFI2 diseases and the
corresponding use of Fast for the production of a drug for the treatment of
leishmanioses, AIDS, listerioses or.infections with other intracellular viral,
bacterial or protozoological pathogens.
In TH1/TH2 diseases, there is a reduced TH1 response. For the
immunological defense, especially in the case of infections with intracellular
pathogens, an effective inflammatory TH1 cell response is decisive.
According to the invention, by the administration of Fast, the immune
CA 02269034 1999-04-16
7
response of the TH1 cells is compensated, which, in the case of diseases
such as leishmaniosis, AmS, listeriosis, trypanosomiasis, bortedella,
leprosy, etc., can combat effectively the intracellularly residing pathogens
of
viral, bacterial or protozoological origin. The addition of Fast thus
compensates a lacking or insu~cient TH1 immune response. Thus, the use
of Fast is disclosed for use as a drug in TH1/TH2 diseases.
The gene product of the human Fast gene sequence is used as therapeutic
agent for the treatment of TH1/TH2 diseases. Physiologically active gene
products of Fast gene segments with potent apoptosis effect also come into
consideration, for example, gene products shortened at the N- or C-terminal,
or gene products, in which one or several intracistronic gene segments are
lacking. v
The use of Fast for the treatment of TH1/TH2 diseases can be applied in
veterinary medicine for mammals, especially for useful animals, above all
for pigs, cattle and sheep, but also for the domestic animals, such as dogs
and cats, and in human pathology, to support the TH1 immune response to
intracellular pathogens. Among useful animals, especially those mammals
2 0 are to be understood which serve humans in transport tasks or in the
agricultural sector for producing food.
Especially preferred is the use of Fast for the treatment of human
leishmanioses, including both cutaneous as well as visceral and also mucosal
2 5 leishmanioses, which are caused by L. donovani, L. infaruum, L. chagasi,
L.
tropica, L. major, L. braziliensis, L. mexicana. Correspondingly, in the
case of useful animals, the intracellular parasitary Leishmania species can be
combatted by dosage of Fast.
.._..._ _. _._._ ___.. . _ . . _.._ . _._._.. .. ..... CA 02269034 1999-04-16
8
Fast induces apoptosis of the infected cells. An embodiment represents the
use of Fast in the pharmacological form in vivo. In another embodiment, in
vitro, the use of Fast can be, used during external blood purification.
Infected blood cells, that is, differentiated cells of the hematopoietic
system,
especially macrophages, are killed ex vivo by the administration of Fast.
The blood treated with Fast is then returned to the patient. Thus, in this
case, Fast is applied externally, in order to avoid any undesirable side
effects of the administration of Fast.
In a preferred embodiment, Fast is administered topically to the patient.
Topical application means nonsystemic application. The substance according
to the invention is then applied onto the epidermis or dropped into the eye,
nose or ears, so that essentially it does not enter into the blood stream.
Formulations of liquid, semiliquid or semisolid type are suitable for topical
application. They are applied onto the cutaneous or mucosal lesions in a
form that is able to be resorted. For example, cream-like formulations,
salves, pastes, drops or lotions are available. The topical application of
Fast can also be done in the solid form by providing powders.
Salves, pastes or cream formulations contain the active substance as an
aqueous or nonaqueous solution or suspension based on a fat-containing or
nonfat carrier substance.
2 5 In another preferred embodiment, the Fast is applied systemically, using
oral, intravenous, intraperitoneal and intramuscular forms of administration.
The formulation for parenteral application is especially preferred.
CA 02269034 1999-04-16
9
As carrier materials for systemic application, especially those in the
following cited forms of administration come into consideration (Hartke &
Mutschler, Deutsches Arzneibuch, 9th Edition, 1986, Volume 3
"Parenteralia", p. 2670-2677, Wissenschaftliche Verlagsgesellschaft
Stuttgart, Govi-Verlag GmbH Frankfurt; Phalnacopoeia Helvetica, Edition
Septima 1/1/1991 and the revised version of 1996, Monograph
"Parenteralia", US Pharmacopeia, National Formulary 18, 1995, p. 1650-
1652).
Depending on the type of application (especially depending on the systemic
or topical application, application in vivo or ex vivo), on the adjuvants of
the
particular galenic preparation, on the infection type, on the infection stage
and on the current course of infection as well as depending on the known
clinical parameters, for example, on the patient's age and the specific
anamnesis of the patient, Fast is administered to the patient at a dose
between 1 picogram and 100 mg per kg of body weight (of the patient).
Application at a concentration between 10-$ and 10-' g/kg of body weight is
preferred.
2 0 In an especially preferred embodiment, the active Fast substance is
combined with other pharmacological carrier materials, which, for example,
improve the phartnacokinetic properties, while at the same are also
physiologically compatible.
2 5 Human Fast; /CD4+/TH1 cells or cells which express Fast only weakly can
be transfected with Fast. The incorporation of Fast gene material can be
done by lipofection, microinjection, with the aid of viral vectors or by
electroporation. The Fast gene material can be provided with a
preconnected promoter and can be transfected in this form, but also it can be
CA 02269034 1999-04-16
inserted in a targeted manner at the position of the physiological promoter.
Clonogenes, cells transfected by the standard method, which express Fast
stably, are used therapeutically for TH1/TH2 diseases in order to enhance
the TH1 immune response.
5
For this purpose, T-lymphocytes are taken from the infected patients and
those TH1 cells are isolated whose T-cell receptors recognize antigens of the
particular infectious pathogen. These cells are cultivated and transfected
with Fast gene material. The Fast gene material can include both the
10 human Fast gene sequence as well as physiologically active Fast segments
or gene segments which are potent in the apoptosis reaction, which express
an Fast protein shortened at the N- or C-terminal. Stable transfected
clonogenous, autologous cells are then applied again into the patient with
Fast deficiency or suppressed TH1 immune response. In this special
embodiment, the TH1 cells transfected with Fast serve for treating
TH1/TH2 diseases. This covers the same infection spectrum that was
already described above for the treatment with Fast. The transfected,
autologous cells mobilize the FasL/FasR apoptosis system and kill the
infected cells, especially macrophages:
According to the invention, it was found that TH1 lymphocytes can kill
Leishmania-infected macrophages in vitro.
In order to check this ability of TH1 lymphocytes experimentally,
2 5 macrophages infected with Leishmania and activated were cocultivated with
Leishmania-induced TH1 cells in the presence of y-interferon. The TH1
cells were taken from the lymph nodes of C57BL/6 mice, which were
previously infected with Leishmania major for 5 to 8 weeks,- by removing
the lymph nodes which take care of the lesions, and obtaining from them the
CA 02269034 1999-04-16
11
TH1 cells, purified by the MACS method (Miltenyi Biotech GmbH,
Bergisch-Gladbach). This material was restimulated in vitro by
promastigotes of L. major (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 served as a source of
antigen-presenting cells (APC). The TH1 cell type was confirmed by
measurement of the y-interferon and interleukin-2 (IL-2) activity. After 72
h, the CD4+ "blast" cells ("blast" cells are those cells which, after
stimulation, went through a clonal expansion) were isolated and washed.
They were cocultivated with bone marrow macrophages, which were
previously infected with L. major in a ratio of 1:1 and in the presence of y-
interferon (SO U/mL). It was found that after cocultivation for 6 hours,
60 % of the macrophages from the C57BL/6 strain were apoptotic. On the
other hand, those macrophages which were treated analogously but which
were taken from the syngenic FasR deficient raised mouse strain lpr showed
almost no apoptotic characteristics.
In order to check the function of the FasL/FasR-controlled apoptosis signal
more thoroughly, moreover, according to the invention, in vivo experiments
2 0 undertaken with the bred mouse strains C57BL/6 (resistant strain) and the
syngenic mouse strains gld (Fast: ) and lpr (FasR ), all of which were mouse
strains gld (FasI: ) and lpr (FasR ), all of which with Leishmania infected
subcutaneously with Leishmania major promastigotes (in the stationary
phase) subcutaneously at a dosage of 2 x 106 (in an end volume of 50 ~cL).
In all three of the above strains, the development of the Leishmania infection
was followed once a week with the aid of the size of the lesions in the paw
of the mouse and measured with a ruler, comparing the size of the infected
paw with the size of the opposite, not infected paw.
CA 02269034 1999-04-16
12
The present invention concerns a material composition containing Fast and
one or several cytokines, especially y-interferon, especially such a material
composition as a drug, especially for the in vivo treatment. The use of the
corresponding material composition according to the invention for the
treatment of TH1/TH2 diseases is claimed, for example, for use of the
treatment of leishmanioses, AIDS, listerioses or infections with other
intracellular viral, bacterial or protozoological pathogens or in a preferred
form for the treatment of cutaneous, visceral or mucosal leishmanioses.
The invention is concerned with the use of autologous CD4+/FasL~/TH1 cell
lines of patients which were transfected with the Fast gene sequence or with
physiologically active segments of the Fast gene sequence for the treatment
of TH1/TH2 diseases, for example, with the use of CD4+/FasL-/TH1 cell
lines transfected with Fast for the treatment of leishmanioses, AIDS,
listerioses or infections with other intracellular, viral, bacterial or
protozoological pathogens, whereby especially the use of CD4+/FasI:/THl
cell lines transfected with Fast is claimed for the treatment of cutaneous,
visceral or mucosal leishmanioses.
2 0 The invention is explained in more detail with the aid of the following
figures.
In Figure 1, it is shown that the resistant control strain CS?BL/6 showed
almost no sign of lesions any longer 12 weeks after the infection event, as
2 5 expected. On the other hand, it is also clear from Figure 1 that a
progressive increase of the size of lesions is observed in the two other mouse
strains, Ipr and gld. This experiment underlines the importance of functional
Fast and FasR for an effective inflammatory TH1 effector function. The
CA 02269034 1999-04-16
13
axes show on the abscissa the number of weeks after the infection event and,
on the ordinate, the size of the lesion in mm.
In Figure 2, it is shown with the aid of dilution series, that the number of
Leishmania parasites is significantly increased in the lesions of lpr or gld
mice in comparison to samples from the lesions of the mouse control group
which has a functioning FasL/FasR signal transfer system (C57BL/6). The
number of parasites per mg of tissue is plotted on the abscissa. For each of
the three mouse strains (with the exception of lpr mice), the number of
parasites was determined after 30 and 90 days. For the lpr mice, the number
of parasites was not determined after 90 days (ND).
It is shown in Figure 3 that, after an in vitro restimulation of the TH1
cells,
a comparable production of y-interferon can be detected in all three mouse
strains (control group, lpr and glc~.
On the other hand, IL-4 (not shown in the Figures), which is characteristic
for TH2 cells, is almost undetectable. Thus it is proven experimentally that
both mutant strains have wild-type properties with regard to cell
proliferation
2 0 and cytokine. production. Moreover, this experiment shows that, in the
mutant strains, there is adequate cytokine production for the initiation of a
TH1 immune response.
Figure 4 shows that potential influences of the immunologically active
CD8+-T-cells with their characteristic cytotoxic effector function can be
excluded by perforin secretion in case of Leishmania infection. A perforin-
deficient mouse strain (knock-out, ko) shows the same kinetic profile in the
elimination of the infectious pathogen as the wild-type strains. The axes
CA 02269034 1999-04-16
14
show the number of weeks after the infection event on the abscissa and the
size of lesion in mm on the ordinate.
Figure S shows the size of the lesions as a function of time after the
original
Leishmania infection with L. major in gld mice, which are known to be
Fast-deficient, (1) after the administration of Fast in the form of a
supernatant of Fast-expressing cell cultures, (2) without administration of
Fast and (3) in mice of the control strain C57BL/6, which did not receive
any Fast injections either. The number of weeks after the infection is
1 o shown on the abscissa and the size of the lesion in mm on the ordinate.
For
this purpose, gld mice were infected with L. major (5 weeks of duration of
infection) and then in case (1), in the subsequent three weeks were injected
with 50 ~.L of a 5-fold concentrated solution of the supernatant of FasL-
produced neuro-2a-cell cultures every four days. In case (1), the FasL-
containing solution was injected into the lesions.
Those gld mice which did not receive any Fast injection show an average
lesion size of 1.3 mm nine weeks after the infection. On the other hand, the
wild-type mice with functional Fast and FasR production, even without the
2 0 addition of Fast, show only an average lesion size of 0.3 mm after the
same
time period. Mice of the gld strain, which received injections with Fast
samples show a clearly reduced lesion size in comparison to the untreated
gld mouse, with an average size of 0.75 mm.
2 5 In Figure 6, the number of parasites per mg tissue is shown on the
abscissa
for the mouse strain gld, treated with Fast or with a control sample, and for
the control strain C57BL/6. The result of this experiment is comparable
with the results from Figure 5. Seven days after the last injection of Fast
samples or control samples from supernatants of pseudotransfected neuro-2a-
CA 02269034 1999-04-16
cells in the gld mouse, more than 40 parasites were detected in 1 mg tissue
for the mice treated with Fast' control samples. Only approximately eight
parasites/mg of tissue were detected in the gld mice treated with Fast. The
control group of the resistant C57BL/6 mice had almost no parasites in their
5 lesions 63 days after the infection event.
The results of these experiments, shown in Figures 5 and 6, can be
summarized by saying that only a cooperation of functional FasR with
functional Fast genes or their functional expression products ensures
10 effective immunological defense against Leishmania infection based on the
TH1 reaction.
It 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.
15 Fxp. Med. 181, 1995), that y-interferon (IFN-y) is of decisive importance
for the effectiveness of the immune response in leishmanioses. Mouse
strains with genetically resistant disposition to leishmanioses show an
insignificant course of the infection as long as they do not have a functional
IFN-'y 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
Buchmuller-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-y is attributed to the induction of NO synthase, which
increases NO synthesis.
CA 02269034 1999-04-16
16
According to the invention, it was found that IFN-y promotes the expression
of FasR on macrophages infected with Leishmania.
It is shown in Figure 7 that the expression of FasR both in the wild-type as
well as in gld mice is increased after the administration of IFN-y. On the
other hand, this effect is not detectable in the FasR deficient lpr mice. Bone
marrow macrophages were taken from six-week old mice of the strain Bone
marrow macrophages were taken from six-week old mice of the strain
C57BL/6, and were cultured according to the data 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). After that, the
macrophages were infected with promastigotes of Leishmania in a ratio of
5:1, washed after 12 hours and were cultured again in the presence of IFN-y
(50 U/mL) for another 48 h in the culture. After marking with monoclonal
anti-Macl antibodies M1/70, 98 % of the macrophages gave a positive
signal. Moreover, the macrophages were marked with a control antibody, a
biotinylated anti-Fas antibody (J02, Pharmingen, USA) or with a
biotinylated monoclonal anti-MHC-II antibody (monoclonal Ak BP10~ and
then fluorescing Streptavidin (Caltag, USA) was added. The analysis was
2 o carried out on an FACScanr flow-through cytometer (Becton Dickinson,
Mountain View, CA, USA).
Furthermore, according to the invention, it was shown that the activation of
the expression of FasR on the macrophages produced by IFN-y led to a
2 5 higher sensitivity of the cells to an Fast-controlled apoptosis. The three
mouse strains named above were used for the experimental detection of IFN-
y-activated macrophages infected with Leishmania. As was described in the
previous paragraph, these cells were raised for 48 h in the culture and then
treated with the supernatant of recombinant Fast-producing neuro-2a cells or
CA 02269034 1999-04-16
17
pseudotransfected neuro-2a cells (Rensing-Ehl, A. , Frei, K. , Flury, F. ,
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 determined with the aid of subdiploid DNA according to the method of
Renno et al. (Renno, T., Hahne, M., Tschopp, J. and MacDonald, H. R., J.
Exp. Med. 183, 431-437, 1996).
Thus, through the FasL/FasR system, TH1 cells can introduce the apoptosis
of macrophages infected with Leishmania or other intracellular pathogens
when and as long as the macrophages were activated first with IFN-'y.
According to the present invention, the IFN-y production for the stimulation
of NO synthesis is not sufficient to explain the experiments on the immune
reactions of mouse strain C57BL/6, gld and lpr toward the Leishmania
pathogens.
It is shown in Figure 8 that the macrophages of the three strains have
approximately comparable rate of synthesis of NO after the addition of IFN-
y. Thus, according to the invention, in addition to the known mechanism of
NO production, the activation of the FasR expression also occurs as a result
2 0 of a corresponding stimulation by IFN-y to combat intracellular infection
in
antigen-presenting cells.
The present invention will be explained in more detail with practical
examples.
First of all, the experimental boundary conditions on which all the following
practical examples were based will be explained.
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The mouse strains C57BL/6 and C3H/HeN were purchased from IFFA-
CREDO (Saint Germain-sur-1'Abresle, France). The two deficient mouse
strains gld (Fast-) and lpr (FasR-) are derived from the strains C57BL/6 or
C3H/HeN and were bred in Jacksons Laboratorium (Bar Harbor, Maine,
USA). All mice were six to eight weeks old at the time of the experiments.
The Leishmania parasites of the strain L. major LV 39 (MRHO/SU/59/P-
strain) were stored in vtvo and raised in vitro according to the method of
Louis, J. A., Moedder, E., Behin, R., Engers, H. D. (Eur. J. Immunol., 9,
l0 841-847, 1979).
The infection of the mice with the parasites was done subcutaneously in one
paw with 2 x 106 promastigotes in the stationary phase with an end volume
of 50 ~.L.
The recombinant Fast was expressed in neuro-2a cells according to 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 determined according to the method of Renno, T.,
Hahne, M., Tschopp, J. and MacDonald, H. R. (J. Exp. Med. 183, 431-
437, 1996) based on the content of subdiploid DNA.
2 5 The bone marrow macrophages originate from bone marrow precursor cells
(after in vitro differentiation) which was carned out according to 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).
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Cultivation of lymphocytes: the cells were taken from those lymph nodes
which were in connection with L. major-produced lesions (3 x 106/mL) and
were stimulated in vitro with UV-irradiated L. major promastigotes (1 x
106/mL) for 72 h or with ConA (2.5 mg/mL) for 48 h on the bottom of
microtiter plates (Falcon, Switzerland). As external conditions, 37°C,
an air
environment enriched to 7 % COz and an end volume of 200 ~.L were
chosen. The cells were cultivated in DMEM (Dulbeccos Modified Eagles
Medium) with 5 % heat-inactivated FCS (Fetal Calf Serum), with L-
glutamine (216 mg/mL), 5 x 10-5 M (i-ME, 10 mM Hepes, 100 UImL of
penicillin and 100 mg/mL of Streptomycin. The supernatants of the
corresponding cultures were combined and stored at -20°C and then they
were used for the cytokine determination. In some experiments, the CD4+-
T cells were removed fiom the cell suspensions by adding, after the
incubation, monoclonal Ig-M antibodies against CD4 marker (RL 172.4) and
Low-Tox rabbit complement (Cedarlane, Homy, Ontario, (RL 172.4) and
Low-Tox rabbit complement (Cedarlane, Horny, Ontario, Canada) according
to the method of 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). In some experiments, the strength of the proliferation was also
2 0 checked by the measurement of the incorporation of 3H-methylthymidine
(3htdr, Amersham, GB).
a)
1. Experimental procedure:
2 5 An in vitro test system was used for the detection of the apoptotic action
of
CD4+/TH1 cells on bone marrow macrophages infected with L. major.
CA 02269034 1999-04-16
T'he CD4+/TH1 cells were taken from mice of the bred strain C57BL/6
(IFFA-CREDO, Saint Germain-sur-1'Abresle, France), which were infected
with L. major 5 to 8 weeks previously.
5 The lymph nodes in the area of the Leishmania lesions were removed and
were purified by the MACS method using the procedure given by the
manufacturer (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany). The
purified CD4+ cells (2 x 105 per well) were stimulated in vitro by
promastigotes of L. major (previously treated with UV irradiation) in the
1 o presence of irradiated (3000 rad) lymph node cell populations without CD4+
cells, so that they can be used as material for antigen-presenting cells
(APC).
After 72 h, the CD4+ "blast" cells (see above) were isolated and washed.
They were cocultivated with bone marrow macrophages which were
previously infected with L. major using the method described above and
15 were activated with y-interferon, in the ratio of 1:1 (in an end volume of
2
mL in Petri dishes). After 6 h of cocultivation, the bone marrow
macrophages were harvested with a plastic scraper, washed and the amount
of apoptotic cells was determined by FACS analysis. For this purpose, the
macrophage population was marked with monoclonal anti-MAC 1 antibodies
2 o M1/70. After marking, 98 % of the macrophages showed a positive signal.
In addition, the macrophages were marked with a control antibody, a
biotinylated anti-Fas antibody (J02, 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 an FACScanr
2 5 flow-through cytometer (Becton Dickinson, Mountain View, CA, USA).
2. Results
It was found that after 6 h of cocultivation, more than 60 % of the
macrophages from the C57BL/6 strain were apoptotic. On the other hand,
CA 02269034 1999-04-16
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those macrophages which were treated analogously but were taken from the
syngenic FasR-deficient mouse strain lpr, showed almost no apoptotic
characteristics (approximately 2 % of the cells).
b)
1. Experimental procedure
The proof, that a TH1/TH2 disease can be treated effectively with Fast, was
provided for various mouse strains infected with L. major. For this purpose,
gld mice, which were infected 5 weeks previously with L. major, were
treated for three weeks every 4 days with a fivefold concentrated supernatant
of the Fast-expressing neuro-2a cells. For this purpose, 50 ~L was injected
into the Leishmania lesions. The size in the lesion was determined during
the entire experiment over nine weeks. The size of the lesions was
determined with the aid of a ruler which permitted measurement of the
thickness of the infected paw in comparison to the thickness of the opposite,
noninfected paw. Control mice, which were also infected, were treated with
the supernatant of neuro-2a pseudotransfectants, which do not express any
Fast (Rensing-Ehl, A. , Frei, K. , Flury, R. , Matiba, B. , Mariani, S . M. ,
Weller, M., Aebischer, P., Kxxmmer, P. H. and Fontana, A.; Eur. J.
2 0 Immunol. 25, 2253-2258, 1995). Seven days after the end of the treatment
(or nine weeks after the infection), the size of the lesions and the number of
parasites in the lesions were determined in gld mice treated in this way. The
number of parasites in the lesions on the infected paws was determined with
a dilution test at certain intervals according to the method of Titus, R. G.,
Marchand, M., Boon, T. and Louis, J. A. (Parasite Immunology 7, 545-
555, 1985).
2. Results
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The result of the treatment of Fast-deficient gld mice with Fast is shown in
Figures 5 and 6. Figure 5 shows the size of the lesion as a function of time
(in weeks, after the infection). The three curves symbolize the following:
1. (i) gld mice, which were treated with the Fast of Fast-expressing cells
for five weeks after the infection; 2. (D) gld mice, which were treated with
the supernatant of Fast pseudotransfectants, and 3. (O) control mice of
strain C57BL/6.
Five weeks after the infection and before the treatment with the various
supernatants, the gld mice showed a lesion size of approximately 0.9 mm.
During the further observation period, the lesion size of the gld mice, into
which no functional Fast was injected, increased (to approximately 1.2 mm
nine weeks after the infection), while the size of the lesion was reduced in
the mice which were treated with functional Fast. Nine weeks after the
infection, these mice show only an average lesion size of 0.6 mm.
This experiment documents that the treatment of Fast-deficient gld mice
with Fast has a significant therapeutic effect on the course of the infection.
2 0 Figure 6 shows the number of parasites per mg of tissue, determined in a
dilution test, in the lesions of C57BL/6 mice, of the Fast treated gld mice
and of gld mice treated with the supernatant of pseudotransfectants per mg of
tissue. The determination of the number of parasites was done nine weeks
after the infection and seven days after the end of the treatment.
The number of parasites is clearly reduced by the injection of Fast (less than
10 parasites per mg of tissue). On the other hand, the mice which were not
treated with Fast show more than 40 parasites per mg of tissue. Thus, this
experimental arrangement also demonstrates clearly the therapeutic effect of
CA 02269034 1999-04-16
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Fast in the treatment of TH1/TH2 diseases, here on the example of
Leishmania infection.
c)
1. Experimental procedure
In order to describe the dependence of the mechanism of apoptosis of
intracellularly infected cells on the presence of y-interferon and thus to
document the activity of y-interferon on the Fas mechanism, bone marrow
macrophages infected with L. major were treated with IFN-'y and finally
investigated for the expression of the corresponding marker proteins.
For this purpose, bone marrow cells of C57BL/6-, gld and lpr mice, where
the gld and lpr mice had a C57BL/6 origin, were cultivated in 10 mL Petri
dishes made of plastic (Sterilin, Hounslow, GB) in DMEM (see above) with
the addition of 20 % horse serum and 30 % serum-free supernatant of L929
cells, as M-CSF donors. After seven days of cultivation, the macrophages
were separated and suspended in DMEM with 10 % FCS (see above), L-
glutamine (216 mg/mL), 10 mM Hepes, 100 U/mL of penicillin and 100
mg/mL of streptomycin. Then the macrophages were infected with L. major
2 0 promastigotes in a ratio of 5:1 and were taken up in an end volume of 2 mL
in Petri dishes made of plastic. Twelve hours after the infection, the bone
marrow macrophages were washed and were incubated in the medium
described above for 48 h with recombinant IFN-y (50 U/mL). During the
last four hours of incubation, the supernatant of either Fast-producing
2 5 neuro-2a cells or neuro-2a pseudotransfectants (20 volume % ) were added
to
the macrophage cultures. Finally, in the macrophages, using the FACS
method described above (see under a)), the cell surface expression of MHC-
II (main histocompatibility complex of class II) and FasR molecules.
CA 02269034 1999-04-16
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2. Results
Figure 7 shows the result of this experiment by plotting the number of cells
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 proven
by the increased expression of MFiC-II molecules. In the three strains, the
integral of the number of cells and the number of expressed MHC-II
molecules is comparable. On the other hand, the number of expressed FasR
molecules as a result of the treatment with IFN-y shows characteristic
differences in the individual strains. Both the wild-type strain as well as
the
gld strain show an increased rate of expression but not the FasR-deficient gld
strain.
Thus, it becomes clear that the treatment with IFN-Y has a direct influence
on the FasL/FasR controlled apoptosis signal by up-regulation of the
expression rate of the FasR molecules by IFN-y.
