Clin. exp. Immunol. (1990) 81, 195-199
Depletion of CD8 + T cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma) production in infected rats M. BAKHIET, T. OLSSON, P. VAN DER MEIDE* & K. KRISTENSSONt Departments of Neurology and *Cellular Neuropathology, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden, and tTNO Primate Centre, Rijswiik, The Netherlands
(Acceptedfor publication 14 February 1990) SUMMARY Sprague-Dawley rats infected with Trypanosoma brucei brucei showed a strong and rapid induction of splenocyte IFN-y (within 12 h post-infection) as measured by a single cell assay for IFN-y secretion. Depletion of CD8+ cells in infected rats abrogated the IFN-y production, suppressed parasite growth and increased survival of the animals. Induction of MHC class I antigens in the paraventricular and supra-optic hypothalamic nuclei caused by the trypanosome infection was also inhibited by the CD8+ cell depletion. It is suggested that the CD8+ cells are involved directly or indirectly in growth regulation of the parasite and that IFN-y induced by the parasite may be one of the factors that trigger MHC expression and immunosuppression.
Keywords MHC class I antigens immunosuppression cytokines
neurons
INTRODUCTION
MATERIALS AND METHODS
Experimental infection with Trypanosoma brucei brucei in the rat induces MHC class I antigens in the paraventricular (PVN) and supra-optic (SO) hypothalamic nuclei in the brain (Schultzberg et al., 1989). The parasites do not seem to penetrate early through the blood-brain barrier, but they localize to areas in the nervous system lacking such a barrier, which include the circumventricular organs (Schultzberg et al., 1988). Parasites are present both in the neurohypophysis and in the median eminence, and in these regions there is also an infiltration of macrophages and T cells. The CD8+ cytotoxic/suppressor cells dominate among the T cells. Hypothetically, the MHC induction may be due to some factor, derived either from the parasites or from the inflammatory cells, interacting with the axon terminals of the PVN and SO which project to the median eminence and the neurohypophysis. To study any role of the CD8 + cells in the pathogenesis of the disease, experiments were carried out to deplete these cells. Since interferon-gamma (IFN-y) is a most potent cytokine to induce MHC antigens (Rosa & Fellous, 1984; Wong et al., 1984; Skoskiewicz et al., 1985), the cellular production of this cytokine was determined during the course of infection. Here we report that trypanosomes in the rat very rapidly trigger IFN-y production and that, unexpectedly, in vivo depletion of CD8+ cells leads to a marked suppression of parasite growth. Correspondence: Dr T. Olsson, Department of Neurology, Huddinge Hospital, Huddinge, S-141 86 Sweden.
Parasites A strain of Trypanosoma brucei brucei was used. The strain, variable antigen type An Tat 1/1, derived from stabilate EATRO 11 25, was a generous gift from Dr Nestor van Meirvenne, Laboratory of Serology, Institute of Tropical Medicine Prins Leopold, Antwerp, Belgium, and was passaged once in rats before use.
Animals and infection procedure Ninety-two male, Sprague-Dawley rats (Alab, Stockholm) aged 2 months and weighing about 200 g were infected with trypanosomes. Each animal was injected intraperitoneally with 0-2 ml of a suspension of trypanosomes in a phosphate saline/ glucose buffer, pH 8 0, containing about 3000-5000 parasites/ pl. Every second or third day the number of parasites in the blood was estimated, the animals weighted and the clinical condition assessed. Thirty-two uninfected rats were used as controls. CD8+ lymphocyte depletion To deplete CD8+ T cells, 0-5 mg OX8 antibody was injected intraperitoneally into 40 rats at the time of infection. The injection was repeated 5 days later. The antibody was prepared from culture supernatants (Holmdahl et al., 1985) of the OX8 hybridoma, originally obtained from Dr A. Williams, Oxford, England. This procedure leads to a complete CD8+ depletion
195
M. Bakhiet et al.
196
Table 1. Antibody specificity and immunoglobulin isotype
Antibodies* DB-1 Rabbit polyclonal OX18 OX8
Specificity
Isotype
Reference
Mouse and rat IFN-y Rat IFN-y MHC class I antigens T suppressor/cytotoxic cells
IgGI IgG2
Van der Meide et al., 1986 Van der Meide et al., 1986 Fukumoto et al., 1982 Brideau et al., 1980
IgGI IgGI
*The hybridoma producing OX8 was from Dr Alan Williams (Oxford, UK); OX8 and DB- 1 were purified from culture supernatants (Holmdahl et al., 1985), OX 18 was purchased from Seralab (Crawley-Down,UK).
100 us
12
0
10
c
E
a
m
90
80 0c 'a
8
ID 4-
6 4
70 60
50 40
I'l
2
V) 6 z)
10
30
20
20
30 40 Time after infection (days)
Fig. 1. Number of surviving rats during infection with Trypanosoma brucei brucei. (- ) Rats in which CD8+ T cells were depleted with OX8; ( ) non-treated rats.
10 I
7
14 21 28 Time after infection (days)
35
Fig. 2. Number of trypanosomes in peripheral blood (PB) during the of infection. A, Anti-CD8 + -treated rats; *, untreated rats. Mean
course
for 14 days. In order to get a more long-lasting lymphocyte depletion, an attempt was made to perform thymectomy 15 days before infection and monoclonal antibody depletion.
Single cell assay for IFN-y production Principally, the method described by Czerkinsky et al. (1988) was used. Nitrocellulose-bottomed, 96-well microtitre plates (Millipore, Bedford, MA) were coated overnight with 100-pl aliquots of the mouse monoclonal antibody DB1 15 pg/ml. All antibodies and their specificities used are listed in Table 1. After repeated washings with PBS, 2% bovine serum albumin was applied for 2-4 h, the plates were washed in PBS and mononuclear cell suspensions were applied followed by incubation overnight at 370C in a humidified atmosphere of 7% CO2. Cells were then removed by flicking the plate, followed by repeated washings in PBS. Polyclonal rabbit anti-rat IFN-y, diluted 1/1000, was applied for 4 h. After washing, biotinylated goat anti-rabbit IgG (Vector Lab, Burlingame, CA) was applied for 4 h followed by avidine-biotine-peroxidase complex (ABC vectastain Elite Kit, Vector Lab). Peroxidase staining with 3amino-9-ethylcarbazole and H202 was performed (Kaplow, 1974). Spots corresponding to cells that had secreted IFN-y were counted in a dissection microscope. ,
and s.d.
are
shown.
260 240 220 aJ) 200 180 01) 160 (D 140 120 0 100 80 E 60 E 40 20 0 "I
T
*AI.
7
6h
Preparation of mononuclear cell suspensions Rats were killed and spleens dissected, crushed through a stainless steel meshwork. The cells were washed once in tissue culture medium. The medium consisted of Iscove's modified
12 h
la
§4v
3 i~j
.i-
i 1A
24 h 3days 7 days 14 days 21 days 28 days Time after infection
Fig. 3. Number of IFN-y immunospots per 106 splenocytes. 0, rats treated with anti-CD81; A, non-treated rats. Stars are uninfected controls. Mean and s.d. are shown.
Effect of CD8+ depletion on T. brucei brucei-infected rats
197
.
Fig. 4. Immunohistochemical staining of CD8+ cells in (a) an untreated rat 14 days post-infection; (b) a CD8+ T cell-depleted rat 14 days post-infection; and (c) a CD8+ T cell-depleted rat 21 days post-infection. Magnification x 85.
Dulbecco's medium (Flow Lab, Irvine, UK) supplemented with 5% fetal calf serum (GIBCO, Paisley, UK), 1% minimal essential medium (Flow Lab), 2 mm glutamine (Flow Lab), 50 yg/ml penicillin, and 60 pl/ml streptomycin. Erythrocytes in the cell pellets were haemolysed by adding 2 ml cold sterile water for 30 sec followed by addition of t ml 2 7% saline. The cells were then washed in medium twice and rediluted to obtain a cell concentration 5 x 106/ml and 100-yl aliquots were then applied to individual microtitre wells in triplicate.
Immunohistochemical technique The rats were killed and the brains, the neurohypophyses and spleens were dissected and snap-frozen. Cryostat sections (8-prm thick) were cut, air-dried and fixed in 2% buffered formaldehyde for 30 sec followed by cold acetone (-20 C) for 30 sec. Sections were blocked in 2% normal horse serum for 30 min and primary monoclonal antibodies (Table 1) were then applied in appropriate dilutions at 40C overnight. After thorough rinsing in PBS biotinylated anti-mouse IgG, cross-absorbed against rat (Vector Lab) and further diluted in 2% normal rat serum, was applied for 30 min. The sections were then incubated with an avidin-biotin-peroxidase complex (Vector Lab.). Controls were sections where the primary antibodies had been omitted. Staining specificity was checked on sections of normal rat spleens. The peroxidase activity was then visualized according to Kaplow (1974). RESULTS The infected, non-depleted rats showed no overt clinical signs of disease during the first 2 weeks post-infection, after which they appeared somewhat lethargic, became moribund and by week 5 post-infection most of them had died. The infected CD8+ lymphocyte-depleted rats showed no clinical signs of disease until 4 weeks post-infection and started to die about 18 days later than the untreated rats (Fig. 1). Parasites were found in the blood 3 days post-infection and their numbers increased
markedly after 14 days in the non-treated rats. The CD8+depleted rats consistently showed much lower numbers of parasites in the blood until 28 days post-infection, when the number of parasites started to increase (Fig. 2). The spleen weights of the infected,untreated rats were increased already 3 days post-infection, and at 21 days post-infection the increase was 12-fold. The depleted rats showed an increase in spleen weights that was about half that of the untreated rats. At autopsy small rests of thymic tissue was found in most rats subjected to thymectomy prior to infection. Cellular production of IFN-y by splenocytes remained at low and normal levels 6 h post-infection. Already 12 h post-infection there was a very marked increase in number of IFN-y-producing cells (Fig. 3). The number of such cells was increased throughout the observation period, but with lower levels by day 21 postinfection. The depleted rats showed no increase in number of IFN-y-producing cells up to 21 days post-infection, but at day 28 their number had markedly increased. Immunohistochemical staining for CD8+ T cells showed a complete loss of labelled cells in the spleen of the depleted rats 7 and 14 days post-infection, but at day 21 a moderate number had reappeared. The infected and untreated rats all showed large numbers of CD8+ cells in their spleens (Fig. 4). Sections through the neurohypophysis and the hypothalamic area showed an infiltration of CD8+ T cells in infected, untreated rats. There was also a marked induction of MHC class I immunoreactivity in these areas and in The PVN and SO (Fig. 5) as previously described (Schultzberg et al., 1989). No CD8+ cells or MHC class I immunoreactivity was seen in the depleted rats up to 21 days post-infection. DISCUSSION The present study shows that the CD8 + T cells play an important role in the pathogenesis of trypanosoma infections, since the number of parasites in the blood was reduced and the survival time of the infected rats was increased when these cells
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M. Bakhiet et al.
Dl~~~~~~~~
Fig. 5. Immunohistochemical staining for MHC class I antigens shows a strong induction in the paraventricular hypothalamic nuclei 14 days post-infection (a); while in a CD8 + T cell-depleted rat no such an induction is seen (b); CD8 + T cells infiltrate the neurohypophysis 14 days post-infection (c); and in a CD8+ T cell-depleted rat these cells are absent (d); Magnification: (a), (b), x 140; (c), (d), x 80.
had been depleted. Injection of the anti-CD8+ cell monoclonal antibody in rats, as performed in our study, causes a complete elimination of CD8 + cells within I h and the effect lasts for 14 days. The thymus will then repopulate the peripheral lymphoid organs with CD8+ cells (Holmdahl et al., 1985). In our study CD8+ cells also reappeared 14-21 days after treatment. This correlates well with the observed effects on the depleted animals, which survived about 18 days longer and showed a delay in the appearance of a marked parasitemia with about 14 days. T. brucei brucei is an extracellular parasite where the effects of the humoral immune response to the variable surface antigens has been the main subject for study and little is known about any role of cytotoxic T cells in the pathogenesis of the disease. This contrasts the situation for the intracellular parasites of the Leishmania species,where the T cells that mediate delayed-type hypersensitivity are involved in a protective response (Heinzel et al., 1989). The presently obtained results with a reduced growth of parasites in CD8+-depleted rats is somewhat unexpected and may imply that this subpopulation of T
cells in some way may promote proliferation of the parasite. Hypothetically, this could be due to some factor released from the activated T cell that directly or indirectly promotes parasite growth; or to an immunosuppression of the host caused either by T cells inducing a state of useless hyper-reactivity of the host that may facilitate the escape of the parasite-specific antigen from the immune defence (Minoprio et al., 1986); or by other factors (Murray et al., 1975; Askonas & Bancroft, 1984). Although evidence has suggested IFN-y to be a macrophageactivating factor that mediates cellular defence to intracellular infectious agents (Nathan et al., 1983; Murray, Rubin & Rothermel, 1983) it can also inhibit macrophage-mediated antigen-specific T cell proliferation (McKernan et al., 1988) and the type of humoral or cellular immune response may be dependent on the timing of IFN-y administration (McKernan et al., 1988). It is therefore interesting that in the present study there was a great increase in the number of IFN-y-producing cells. This response came remarkably early with a very large number of IFN-y-producing cells already 12 h post-infection,
Effect of CD8+ depletion on T. brucei brucei-infected rats which is much earlier than the rise in serum IFN a, /3 and y observed by Bancroft et al. (1983), where it could be measured only when overt parasitaemia had appeared. The increase in IFN-y production was largely abrogated by the CD8+ cell depletion, suggesting that this subset of T cells is mainly responsible for the IFN-y production. Our study indicates an interplay between the trypanosome proliferation and factors derived from activated T cells during the infection. Differences in interplay between various strains of trypanosomes and such factors from different host animals may explain why the infection sometimes results in complete resistance, sometimes in tolerance with a low grade of parasite growth and sometimes in disease with a high level of parasite proliferation. As described in our previous study, there was consistently a strong MHC class I expression in the PVN and SO in the hypothalamus. Hypothetically, this MHC induction may be the result of a factor interacting with the axon terminals of these neurons, which projects to the median eminence and neurohypophysis (Schultzberg et al., 1989). These areas lack a bloodbrain barrier and the axons are therefore exposed to factors in the serum. Since IFN-y is a most potent inducer of MHC antigens (Skoskiewicz et al., 1985) this factor may also be responsible for this reaction in the central nervous system during trypanosome infections. In fact, the CD8+-depleted rats with low production of IFN-y showed no MHC immunoreactivity in the PVN and SO. However, other cytokines and factors induced by the trypanosome infection (Askonas & Bancroft, 1984) may also be involved in this response and in relation to the pathogenesis of clinical signs of African sleeping sickness it is interesting to note that immunomodulatory factors may alter the endocrine secretion from the hypothalamic nuclei as well as the cortical EEG activity (Berkenbosch et al., 1987; Kidron et al., 1989).
ACKNOWLEDGMENTS This investigation received financial assistance from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. The expert technical assistance by Eva-Britt Samuelsson is gratefully acknowledged.
REFERENCES ASKONAS, A.B. & BANCROFT, G.J. (1984) Interaction of African trypanosomes with the immune system. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 307, 41. BANCROFT, G.J., SUTTON, C.J., MORRIS, A.G. & ASKONAS, B.A. (1983) Production of interferons during experimental African trypanosomiasis. Clin. exp. Immunol. 52, 135. BERKENBOSCH, F., VAN OERS, J., DEL REY, A., TILDERS, F. & BESEDOVSKY, H. (1987) Cortiotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science, 238, 524. BRIDEAU, R.J., CARTER, P.B., MACMASTER, W.R. & WILLIAMS, A.F. (1980) Two subsets of rat T-lymphocytes defined with monoclonal antibodies. Eur. J. Immunol. 10, 609. CZERKINSKY, C., ANDERSSON, G., EKRE, H-P, NILSSON, L-A., KLARESKOG, L. & OUCHTERLONY, 0. (1988) Reverse ELI-SPOT assay for
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clonal analysis of cytokine production. I. Enumeration of gammainterferon-secreting cells. J immunol. Methods, 110, 29. FUKUMOTO, T., MACMASTER, W.R. & WILLIAMS, A.F. (1982) Mouse monoclonal antibodies against rat major histocompatibility antigens. Two I a antigens and expression of 1 a and class 1 antigens in rat thymus. Eur. J. Immunol. 12, 174. HEINZEL, F.P., SADICK, M.D., HOLADAY, B.J., COFFMAN, R.L. & LOCKSLEY, R.M. (1989) Reciprocal expression of interferon or interleukin 4 during the resolution or progression of murine Leishmaniasis. Evidence for expansion of distinct helper T cell subsets. J. exp. Med. 169, 59. HOLMDAHL, R., OLSSON, T., MORAN, T. & KLARESKOG, L. (1985) In vivo treatment of rats with monoclonal anti-T-cell antibodies. Immunohistochemical and functional analysis in normal rats and experimental allergic neuritis. Scand. J. Immunol. 22, 157. KAPLOW, L.S. (1974) Substitute for benzidine in myeloperoxidase stains. Am. J. clin. Pathol. 63, 451. KIDRON, D., SAPHIER, D., OVADIA, H., WEIDENFELD, J. & ABRAMSKY, 0. (1989) Central administration of immunomodulatory factors alters neural activities and adrenocortical secretion. Neurol. Ind. 37, (Suppl.) 225. McKERNAN, L.N., BLANK, K.J., SPITALNY, G.L. & MURASKO, D.-M. (1988) Inhibition of macrophage-induced antigen specific T-cell proliferation by interferon-y. Cell. Immunol. 114, 432. MINOPRIO, P.M., COUTINHO, A., JOSKOWICZ, M., IMPERIO, D., LIMA, M.R. & EISEN, H. (1986) Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. II. cytotoxic T lymphocytes. Scand. J. Immunol. 24, 669. MURRAY, H.W., RUBIN, B.Y. & ROTHERMEL, C.D. (1983) Killing of intracellular Leishmania donovani by lymphokine-stimulated human mononuclear phagocytes. Evidence that interferon-y is the activating lymphokine. J. clin. Invest. 72, 1506. MURRAY, P.K., JENNINGS, F.W., MURRAY, M. & URQUHART, G.M. (1975) the nature of immunosuppression in Trypanosoma brucei infections in mice. Immunology, 27, 815. NATHAN, C.F., MURRAY, H.W., WIEBE, M.E. & RUBIN, B.Y. (1983) Identification of interferon-y as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. exp. Med. 158, 670. ROSA, F. & FELLOUS, M. (1984) The effect of gamma interferon on MHC antigens. Immunol. Today, 5, 261. SCHULTZBERG, M., AMBATSIS, M., SAMUELSSON, E.-B., KRISTENSSON, K., VAN MEIRVENNE, N. (1988) Spread of Trypanosoma brucei to the nervous system: early attack on circumventricular organs and sensory ganglia. J. Neurosci. Res. 21, 56. SCHULTZBERG, M., OLSSON, T., SAMUELSSON, E.-B., MAEHLAEN, J. & KRISTENSSON, K. (1989) Early major histocompatibility complex (MHC) class I antigen induction in hypothalamic supraoptic and paraventricular nuclei in trypanosome-infected rats. J. Neuroimmunol. 24, 105. SKOSKIEWICZ, M.J., CALVIN, R.B., SCHNEEBERGER, E.E. & RUSSEL, P.S. (1985) Widespread and selective induction of major histocompatibility complex determined antigens in vivo by gamma interferon. J. exp. Med. 162, 1645. VAN DER MEIDE, P.H., DUBBELD, M., VIJVERBERG, K., Kos, T., SCHELLEKENS, H. (1986) The purification and characterization of rat gamma interferon by use of two monoclonal antibodies. J. gen. Virol. 67, 1059. WONG, G.H.W., BARTLETT, P.F., CLARK-LEWIS, S., BALTYE, F. & SCHRADER,J.W. (1984) Inducible expression of H-2 and Ia antigen on brain cells. Nature, 310, 688.
Depletion of CD8 + T cells suppresses growth of Trypanosoma brucei brucei and interferon-gamma) production in infected rats M. BAKHIET, T. OLSSON, P. VAN DER MEIDE* & K. KRISTENSSONt Departments of Neurology and *Cellular Neuropathology, Karolinska Institute, Huddinge Hospital, Huddinge, Sweden, and tTNO Primate Centre, Rijswiik, The Netherlands
(Acceptedfor publication 14 February 1990) SUMMARY Sprague-Dawley rats infected with Trypanosoma brucei brucei showed a strong and rapid induction of splenocyte IFN-y (within 12 h post-infection) as measured by a single cell assay for IFN-y secretion. Depletion of CD8+ cells in infected rats abrogated the IFN-y production, suppressed parasite growth and increased survival of the animals. Induction of MHC class I antigens in the paraventricular and supra-optic hypothalamic nuclei caused by the trypanosome infection was also inhibited by the CD8+ cell depletion. It is suggested that the CD8+ cells are involved directly or indirectly in growth regulation of the parasite and that IFN-y induced by the parasite may be one of the factors that trigger MHC expression and immunosuppression.
Keywords MHC class I antigens immunosuppression cytokines
neurons
INTRODUCTION
MATERIALS AND METHODS
Experimental infection with Trypanosoma brucei brucei in the rat induces MHC class I antigens in the paraventricular (PVN) and supra-optic (SO) hypothalamic nuclei in the brain (Schultzberg et al., 1989). The parasites do not seem to penetrate early through the blood-brain barrier, but they localize to areas in the nervous system lacking such a barrier, which include the circumventricular organs (Schultzberg et al., 1988). Parasites are present both in the neurohypophysis and in the median eminence, and in these regions there is also an infiltration of macrophages and T cells. The CD8+ cytotoxic/suppressor cells dominate among the T cells. Hypothetically, the MHC induction may be due to some factor, derived either from the parasites or from the inflammatory cells, interacting with the axon terminals of the PVN and SO which project to the median eminence and the neurohypophysis. To study any role of the CD8 + cells in the pathogenesis of the disease, experiments were carried out to deplete these cells. Since interferon-gamma (IFN-y) is a most potent cytokine to induce MHC antigens (Rosa & Fellous, 1984; Wong et al., 1984; Skoskiewicz et al., 1985), the cellular production of this cytokine was determined during the course of infection. Here we report that trypanosomes in the rat very rapidly trigger IFN-y production and that, unexpectedly, in vivo depletion of CD8+ cells leads to a marked suppression of parasite growth. Correspondence: Dr T. Olsson, Department of Neurology, Huddinge Hospital, Huddinge, S-141 86 Sweden.
Parasites A strain of Trypanosoma brucei brucei was used. The strain, variable antigen type An Tat 1/1, derived from stabilate EATRO 11 25, was a generous gift from Dr Nestor van Meirvenne, Laboratory of Serology, Institute of Tropical Medicine Prins Leopold, Antwerp, Belgium, and was passaged once in rats before use.
Animals and infection procedure Ninety-two male, Sprague-Dawley rats (Alab, Stockholm) aged 2 months and weighing about 200 g were infected with trypanosomes. Each animal was injected intraperitoneally with 0-2 ml of a suspension of trypanosomes in a phosphate saline/ glucose buffer, pH 8 0, containing about 3000-5000 parasites/ pl. Every second or third day the number of parasites in the blood was estimated, the animals weighted and the clinical condition assessed. Thirty-two uninfected rats were used as controls. CD8+ lymphocyte depletion To deplete CD8+ T cells, 0-5 mg OX8 antibody was injected intraperitoneally into 40 rats at the time of infection. The injection was repeated 5 days later. The antibody was prepared from culture supernatants (Holmdahl et al., 1985) of the OX8 hybridoma, originally obtained from Dr A. Williams, Oxford, England. This procedure leads to a complete CD8+ depletion
195
M. Bakhiet et al.
196
Table 1. Antibody specificity and immunoglobulin isotype
Antibodies* DB-1 Rabbit polyclonal OX18 OX8
Specificity
Isotype
Reference
Mouse and rat IFN-y Rat IFN-y MHC class I antigens T suppressor/cytotoxic cells
IgGI IgG2
Van der Meide et al., 1986 Van der Meide et al., 1986 Fukumoto et al., 1982 Brideau et al., 1980
IgGI IgGI
*The hybridoma producing OX8 was from Dr Alan Williams (Oxford, UK); OX8 and DB- 1 were purified from culture supernatants (Holmdahl et al., 1985), OX 18 was purchased from Seralab (Crawley-Down,UK).
100 us
12
0
10
c
E
a
m
90
80 0c 'a
8
ID 4-
6 4
70 60
50 40
I'l
2
V) 6 z)
10
30
20
20
30 40 Time after infection (days)
Fig. 1. Number of surviving rats during infection with Trypanosoma brucei brucei. (- ) Rats in which CD8+ T cells were depleted with OX8; ( ) non-treated rats.
10 I
7
14 21 28 Time after infection (days)
35
Fig. 2. Number of trypanosomes in peripheral blood (PB) during the of infection. A, Anti-CD8 + -treated rats; *, untreated rats. Mean
course
for 14 days. In order to get a more long-lasting lymphocyte depletion, an attempt was made to perform thymectomy 15 days before infection and monoclonal antibody depletion.
Single cell assay for IFN-y production Principally, the method described by Czerkinsky et al. (1988) was used. Nitrocellulose-bottomed, 96-well microtitre plates (Millipore, Bedford, MA) were coated overnight with 100-pl aliquots of the mouse monoclonal antibody DB1 15 pg/ml. All antibodies and their specificities used are listed in Table 1. After repeated washings with PBS, 2% bovine serum albumin was applied for 2-4 h, the plates were washed in PBS and mononuclear cell suspensions were applied followed by incubation overnight at 370C in a humidified atmosphere of 7% CO2. Cells were then removed by flicking the plate, followed by repeated washings in PBS. Polyclonal rabbit anti-rat IFN-y, diluted 1/1000, was applied for 4 h. After washing, biotinylated goat anti-rabbit IgG (Vector Lab, Burlingame, CA) was applied for 4 h followed by avidine-biotine-peroxidase complex (ABC vectastain Elite Kit, Vector Lab). Peroxidase staining with 3amino-9-ethylcarbazole and H202 was performed (Kaplow, 1974). Spots corresponding to cells that had secreted IFN-y were counted in a dissection microscope. ,
and s.d.
are
shown.
260 240 220 aJ) 200 180 01) 160 (D 140 120 0 100 80 E 60 E 40 20 0 "I
T
*AI.
7
6h
Preparation of mononuclear cell suspensions Rats were killed and spleens dissected, crushed through a stainless steel meshwork. The cells were washed once in tissue culture medium. The medium consisted of Iscove's modified
12 h
la
§4v
3 i~j
.i-
i 1A
24 h 3days 7 days 14 days 21 days 28 days Time after infection
Fig. 3. Number of IFN-y immunospots per 106 splenocytes. 0, rats treated with anti-CD81; A, non-treated rats. Stars are uninfected controls. Mean and s.d. are shown.
Effect of CD8+ depletion on T. brucei brucei-infected rats
197
.
Fig. 4. Immunohistochemical staining of CD8+ cells in (a) an untreated rat 14 days post-infection; (b) a CD8+ T cell-depleted rat 14 days post-infection; and (c) a CD8+ T cell-depleted rat 21 days post-infection. Magnification x 85.
Dulbecco's medium (Flow Lab, Irvine, UK) supplemented with 5% fetal calf serum (GIBCO, Paisley, UK), 1% minimal essential medium (Flow Lab), 2 mm glutamine (Flow Lab), 50 yg/ml penicillin, and 60 pl/ml streptomycin. Erythrocytes in the cell pellets were haemolysed by adding 2 ml cold sterile water for 30 sec followed by addition of t ml 2 7% saline. The cells were then washed in medium twice and rediluted to obtain a cell concentration 5 x 106/ml and 100-yl aliquots were then applied to individual microtitre wells in triplicate.
Immunohistochemical technique The rats were killed and the brains, the neurohypophyses and spleens were dissected and snap-frozen. Cryostat sections (8-prm thick) were cut, air-dried and fixed in 2% buffered formaldehyde for 30 sec followed by cold acetone (-20 C) for 30 sec. Sections were blocked in 2% normal horse serum for 30 min and primary monoclonal antibodies (Table 1) were then applied in appropriate dilutions at 40C overnight. After thorough rinsing in PBS biotinylated anti-mouse IgG, cross-absorbed against rat (Vector Lab) and further diluted in 2% normal rat serum, was applied for 30 min. The sections were then incubated with an avidin-biotin-peroxidase complex (Vector Lab.). Controls were sections where the primary antibodies had been omitted. Staining specificity was checked on sections of normal rat spleens. The peroxidase activity was then visualized according to Kaplow (1974). RESULTS The infected, non-depleted rats showed no overt clinical signs of disease during the first 2 weeks post-infection, after which they appeared somewhat lethargic, became moribund and by week 5 post-infection most of them had died. The infected CD8+ lymphocyte-depleted rats showed no clinical signs of disease until 4 weeks post-infection and started to die about 18 days later than the untreated rats (Fig. 1). Parasites were found in the blood 3 days post-infection and their numbers increased
markedly after 14 days in the non-treated rats. The CD8+depleted rats consistently showed much lower numbers of parasites in the blood until 28 days post-infection, when the number of parasites started to increase (Fig. 2). The spleen weights of the infected,untreated rats were increased already 3 days post-infection, and at 21 days post-infection the increase was 12-fold. The depleted rats showed an increase in spleen weights that was about half that of the untreated rats. At autopsy small rests of thymic tissue was found in most rats subjected to thymectomy prior to infection. Cellular production of IFN-y by splenocytes remained at low and normal levels 6 h post-infection. Already 12 h post-infection there was a very marked increase in number of IFN-y-producing cells (Fig. 3). The number of such cells was increased throughout the observation period, but with lower levels by day 21 postinfection. The depleted rats showed no increase in number of IFN-y-producing cells up to 21 days post-infection, but at day 28 their number had markedly increased. Immunohistochemical staining for CD8+ T cells showed a complete loss of labelled cells in the spleen of the depleted rats 7 and 14 days post-infection, but at day 21 a moderate number had reappeared. The infected and untreated rats all showed large numbers of CD8+ cells in their spleens (Fig. 4). Sections through the neurohypophysis and the hypothalamic area showed an infiltration of CD8+ T cells in infected, untreated rats. There was also a marked induction of MHC class I immunoreactivity in these areas and in The PVN and SO (Fig. 5) as previously described (Schultzberg et al., 1989). No CD8+ cells or MHC class I immunoreactivity was seen in the depleted rats up to 21 days post-infection. DISCUSSION The present study shows that the CD8 + T cells play an important role in the pathogenesis of trypanosoma infections, since the number of parasites in the blood was reduced and the survival time of the infected rats was increased when these cells
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Fig. 5. Immunohistochemical staining for MHC class I antigens shows a strong induction in the paraventricular hypothalamic nuclei 14 days post-infection (a); while in a CD8 + T cell-depleted rat no such an induction is seen (b); CD8 + T cells infiltrate the neurohypophysis 14 days post-infection (c); and in a CD8+ T cell-depleted rat these cells are absent (d); Magnification: (a), (b), x 140; (c), (d), x 80.
had been depleted. Injection of the anti-CD8+ cell monoclonal antibody in rats, as performed in our study, causes a complete elimination of CD8 + cells within I h and the effect lasts for 14 days. The thymus will then repopulate the peripheral lymphoid organs with CD8+ cells (Holmdahl et al., 1985). In our study CD8+ cells also reappeared 14-21 days after treatment. This correlates well with the observed effects on the depleted animals, which survived about 18 days longer and showed a delay in the appearance of a marked parasitemia with about 14 days. T. brucei brucei is an extracellular parasite where the effects of the humoral immune response to the variable surface antigens has been the main subject for study and little is known about any role of cytotoxic T cells in the pathogenesis of the disease. This contrasts the situation for the intracellular parasites of the Leishmania species,where the T cells that mediate delayed-type hypersensitivity are involved in a protective response (Heinzel et al., 1989). The presently obtained results with a reduced growth of parasites in CD8+-depleted rats is somewhat unexpected and may imply that this subpopulation of T
cells in some way may promote proliferation of the parasite. Hypothetically, this could be due to some factor released from the activated T cell that directly or indirectly promotes parasite growth; or to an immunosuppression of the host caused either by T cells inducing a state of useless hyper-reactivity of the host that may facilitate the escape of the parasite-specific antigen from the immune defence (Minoprio et al., 1986); or by other factors (Murray et al., 1975; Askonas & Bancroft, 1984). Although evidence has suggested IFN-y to be a macrophageactivating factor that mediates cellular defence to intracellular infectious agents (Nathan et al., 1983; Murray, Rubin & Rothermel, 1983) it can also inhibit macrophage-mediated antigen-specific T cell proliferation (McKernan et al., 1988) and the type of humoral or cellular immune response may be dependent on the timing of IFN-y administration (McKernan et al., 1988). It is therefore interesting that in the present study there was a great increase in the number of IFN-y-producing cells. This response came remarkably early with a very large number of IFN-y-producing cells already 12 h post-infection,
Effect of CD8+ depletion on T. brucei brucei-infected rats which is much earlier than the rise in serum IFN a, /3 and y observed by Bancroft et al. (1983), where it could be measured only when overt parasitaemia had appeared. The increase in IFN-y production was largely abrogated by the CD8+ cell depletion, suggesting that this subset of T cells is mainly responsible for the IFN-y production. Our study indicates an interplay between the trypanosome proliferation and factors derived from activated T cells during the infection. Differences in interplay between various strains of trypanosomes and such factors from different host animals may explain why the infection sometimes results in complete resistance, sometimes in tolerance with a low grade of parasite growth and sometimes in disease with a high level of parasite proliferation. As described in our previous study, there was consistently a strong MHC class I expression in the PVN and SO in the hypothalamus. Hypothetically, this MHC induction may be the result of a factor interacting with the axon terminals of these neurons, which projects to the median eminence and neurohypophysis (Schultzberg et al., 1989). These areas lack a bloodbrain barrier and the axons are therefore exposed to factors in the serum. Since IFN-y is a most potent inducer of MHC antigens (Skoskiewicz et al., 1985) this factor may also be responsible for this reaction in the central nervous system during trypanosome infections. In fact, the CD8+-depleted rats with low production of IFN-y showed no MHC immunoreactivity in the PVN and SO. However, other cytokines and factors induced by the trypanosome infection (Askonas & Bancroft, 1984) may also be involved in this response and in relation to the pathogenesis of clinical signs of African sleeping sickness it is interesting to note that immunomodulatory factors may alter the endocrine secretion from the hypothalamic nuclei as well as the cortical EEG activity (Berkenbosch et al., 1987; Kidron et al., 1989).
ACKNOWLEDGMENTS This investigation received financial assistance from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. The expert technical assistance by Eva-Britt Samuelsson is gratefully acknowledged.
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