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Immunological features of HTLV-I myelopathy in Rio de Janeiro, Brazil, and in vitro effects of Cyclosporin A
JOURNAL
OF THE
NEUROLOGICAL SCIENCES
EL-SEVIER
Journal of the Neurological Sciences139(1996) 7- 14
Immunological features of HTLV-I myelopathy in Rio de Janeiro, Brazil, and in vitro effects of Cyclosporin A Maria J. Andrada-Serpa a** , Doris Schor a, Abelardo Q.-C. Aratijo b, Vivian M. Rumjanek ’ a Institute ’ Institute
de Biojkica
National de Cancer. Centro de Pesquisa Ba’sica, Praqa Cruz Vertnelha, 23, 20230-130, Rio de Janeiro, Brazil b Hospital Euandro Chagas, Funda@o Oswald0 Cm z, 21040-360, Rio de Janeiro, Brazil Caries Chagas Filho, Centro de Cikkias da Saiide, Uniuersidade Federal do Rio de Janeiro, Ilha do Fundiio. Rio de Janeiro, Brazil
Received 23 May 1995;revised 24 November 1995;accepted9 December 1995
Abstract Brazilian patients with HTLV-I myelopathy present a significant spontaneous lymphocyte proliferation (SLP), and an increased response to IL-2 exogenous stimulation, in both peripheral blood lymphocytes and in whole blood proliferative assays, when compared to the control group. High antibody titers against HTLV-I antigens were also observed in comparison to healthy seropositive individuals. IL-6 was detected in cerebrospinal fluid (CSF) of 50% of the patients (10 out of 20) and TNF-(Y in four out of nineteen individuals. No correlation was found between the presence of levels of cytokines IL-6 and TNF-a and duration or severity of disease. The addition of cyclosporin A (CsA) significantly inhibited SLP suggesting that this therapeutic agent should be studied in HTLV-1 myelopathy. Brazilian patients with HTLV-I myelopathy present the same immunological abnormalities described in other endemic regions. The whole blood assay reflects the same results of separated blood cells and, due to its rapid execution may be used as an assay to follow clinical trials. Keywords:
HTLV-I myelopathy; Healthy carriers: Spontaneouslymphocytic proliferation; Cyclosporin A inhibition; Interleukin-6; TNF-a
1. Introduction The human T lymphotropic virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL) (Uchiyama et al., 1977; Poiesz et al., 1980; Blattner et al., 1982) and is also associated with a chronic and progressive myelopa-
thy named tropical spastic paraparesis (TSP) or HTLV-Iassociated myelopathy (HAM) (Gessain et al., 1985; Osame et al., 1986). Recently, other diseases were also related to HTLV-I infection (Mochizuki et al., 1992; Vernant et al., 1990; LaGrenade et al., 1990; Tsukasaki et al., 1994; AraGjo et al., 1995). HTLV-1 myelopathy is clinically characterized by a progressive spastic paraparesis with antibodies to HTLV-I in the serum and cerebrospinal fluid (CSF). Although
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HTLV-I has been isolated from CSF lymphocytes, the way through which the virus is related to the pathogenesis of this myelopathy still remains unknown. Several immunological abnormalities have been reported in these patients: (a) a hyperimmune activity with high antibody titers to HTLV-I in serum and cerebrospinal fluid (Osame et al., 19871, (b) the presence of activated lymphocytes in peripheral blood (Itoyama et al., 1988a), (c) the presence of spontaneous lymphocyte proliferation in vitro (Itoyama et al., 1988b; Jacobson et al., 1988) in patients and infected healthy individuals, and (d) a high level of cytotoxic T lymphocytes for HTLV-1 in the peripheral blood and in the cerebrospinal fluid (Jacobson et al., 1990; Jacobson et al., 1992). Furthermore, HTLV-I tax protein transactivates several cytokines and proto-oncogens genes involved in the activation and proliferation of lymphocytes (Siekevitz et al., 1987; Nagata et al., 1989; Tschachler et al., 19891, and a clinical improvement was observed, in some geographic regions with steroids therapy. All this indirect evidence suggests that inflammatory and immunological
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events may play a main role in the pathogenesis of the demyelinating process observed in these patients. Several reports have shown that Brazil is an endemic area for HTLV-I (Andrada-Serpa et al., 1988; AndradaSerpa et al., 1989; Cortes et al., 1989) and cases of ATL as well as HTLV-I myelopathy have already been described (Oliveira et al., 1990; Araujo et al., 1992; Andrada-Serpa et al., 1993; Andrada-Serpa et al., 1995). In the present study, the spontaneous and induced lymphocyte proliferation in Brazilian patients with HTLV- 1 myelopathy and HTLV-I infected healthy individuals was studied, as well as assessing the effect of cyclosporin A (CsA) in such cultures. Immunological activation at the central nervous system was also addressed and interleukin-6 (IL-6) and tumor necrosis alpha factor (TNF-o) quantified in the cerebrospinal fluid. The HTLV-I antibody titer in the patient’s sera was also measured.
2. Materials
and methods
2.1. Patients and serological tests Blood samples were obtained from the following groups: (a) twenty-four patients with HTLV-1 myelopathy; (b) nine HTLV-I seropositive family members; (c) forty-one HTLV-I seronegative family members; (d) five patients with myelopathy HTLV-I negative; and (e) nineteen healthy HTLV-I negative individuals as a control group. Patients had been attending the unit of HTLV neuropathies at the Hospital Evandro Chagas, FIOCRUZ, Rio de Janeiro. The diagnosis of HTLV-I myelopathy was based on the criteria established at the World Health Organization Kagoshima Meeting (Osame, 1990). Sera were screened by indirect immunofluorescence using two infected cell lines MT-2 and C91p1, the ones found positive were confirmed by a commercial western blot p21 enhanced (Cambridge Biotech Corporation, Worcester), the criteria of positivity was the expression of gag and env genes. All the sera from both the patients and the healthy carriers presented a blot pattern (~19 stronger than ~24) compatible with HTLV-I infection (Chen et al., 1990; La1 et al., 1991). The presence of HTLV-I antibody in the CSF patients as well as antibodies titers was performed by indirect immunofluorescence using both infected cell lines. 2.2. Immunological studies 2.2.1. Lymphocyte proliferation Peripheral blood lymphocytes (PBL) were separated from heparinized venous blood by density gradient centrifugation over Ficoll-Hypaque and washed twice in phosphate buffered saline (PBS). One hundred ~1 of the cell suspension (1 X lo6 cells/ml) in RPM1 1640 (Sigma
Company, Saint Louis, USA) supplemented with 2 mM L-glutamine, 100 U/ml of penicillin, 100 pg/ml of streptomycin and 10% of fetal calf serum (FCS) were plated in sterile flat-bottom 96-well microplates (Falcon 3072, Becton Dickinson, Oxnard, CA) and incubated with or without 5 kg/ml of PHA (Sigma Company, Saint Louis, USA), or with or without 10% of IL-2 (Lymphocult-T-LF, Biotest Diagnostics, Germany). Cyclosporin A (Sandoz, Basel, Switzerland) at a concentration of 0.5 or 1 pg/ml as well as exogenous stimulation (PHA and IL-2) were added at the beginning of the cultures. Cultures were maintained at 37°C in an atmosphere of 5% of CO, for three and five days. All cultures were performed in triplicate.
2.2.2. Whole blood proliferation Lymphocyte proliferation using whole blood (Moraes et al., 1989) was performed with blood diluted 5 times in RPM1 1640 without FCS and 100 pl were plated in triplicate with 100 pl of RPM1 1640 supplemented with 2 mM I-glutamine, 100 U/ml of penicillin, 100 p.g/ml of streptomycin and without FCS and incubated with or without 5 pi/ml of PHA or 10% of IL-2. Cyclosporin A at a concentration of 0.5 or 1 kg/ml was added at the beginning of the cultures. The cultures were maintained at 37°C in an atmosphere of 5% of CO, for five days. Lymphocyte proliferation was assessedby the uptake of [3H] thymidine. Cells were pulsed with 0.5 I.&i per well of [ 3H] thymidine (specific activity of 2 Ci/mmol) (Amersham International, Amersham, England, UK) for 6 h, harvested, and the radioactivity counted per minute (cpm) on a liquid scintillation counter (Beckman Instruments, Inc., CA, USA).
2.2.3. Cytokines Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-CY) were quantified in twenty CSF of patients with HTLV- 1 myelopathy. A commercial sandwich enzyme immunoassay (Research and Diagnostics Systems, Minneapolis, MN, USA) was used. Briefly, samples and standards were incubated in anti-IL-6 or anti-TNF-ol monoclonal antibody-coated polystyrene microtitre wells. Cytokine binds to antibody on the coated well, the unbound samples were removed by washing. After washing, an enzyme-linked polyclonal antibody specific for IL-6 or TNF-o was added to the wells to ‘sandwich’ the immobilized cytokine. Following a wash, a substrate solution was added to the wells and color develops in proportion to the amount of cytokine bound in the initial step. The reaction was stopped by the addition of 2N H,SO, and optical density was measured at 450 nm. According to the manufacturer’s instructions these immunoassays are able to detect TNF-o at a concentration greater than 4.8 pg/ml and IL-6 at one greater than 0.35 pg/ml. Standard curves
M.J. Andrada-Serpa et al. /Journal
were obtained with serial dilution of recombinant human TNF-a and of recombinant human IL-6 provided with immunoassays. 2.3. Statistical analysis Spontaneous proliferation and proliferation induced by exogenous stimulus were analyzed by Mann-Whitney Utest. The results obtained with cyclosporin A were evaluated by Wilcoxon’s test for paired data. Student’s t-test was also used to compare serological titers between patients and healthy infected individuals.
3. Results 3. I. Spontaneous lymphocyte proliferation In the whole blood system without exogenous stimulation a significant difference was observed between HTLV- 1 myelopathy patients and the other groups varying from
p < 0.01, when compared to the control group and seronegative relatives, to p < 0.05 when compared to seropositives. On the other hand, no difference was observed between seropositive and negative relatives (Fig. 1A). The results of spontaneous lymphocyte proliferation of PBL after 3 and 5 days of incubation are shown in the Fig. 1B. Patients with HTLV-1 myelopathy presented a significant spontaneous proliferation when compared to the control and seronegative groups (varying from p < 0.01 to p < 0.05). After 3 days of incubation a significant difference ( p < 0.05) was obtained between the patient group and seropositive individuals, but after 5 days of incubation this effect disappeared. No difference was observed between seropositive and negative relatives after 3 and 5 days of incubation. The levels of spontaneous proliferation obtained in our assays were lower than the ones obtained by other authors mainly due to the time interval used for pulse of [ ‘Hlthymidine (6 as opposed to 18 h), and perhaps to the specific activity of [3H]thymidine used in this study. Even with these experimental differences we were able to
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TSP/HAM
Relatives+
Relatives-
Control
TSP/HM
Relatives+
Relatives-
Control
Fig. 1. Spontaneous proliferation: (A) Whole blood: Patients: mean of 421.7 cpm (SD k 3 19.4); Relatives + : mean of 243.2 cpm (SD + 159.9); Relatives - : mean of 206 cpm (SD k 8 1.5); Controls: mean of 175.2 cpm (SD + 53.3). (B) 0 PBL 3 days: Patients: mean of 702.5 cpm (SD + 289.9); Relatives + : mean of 458.8 cpm (SD f 115.5); Relatives - : mean of 503.3 cpm (SD* 129.1); Controls: mean of 506.9 cpm (SD* 162.4). 0 PBL 5 days: Patients: mean of 1225.6 cpm (SD + 608.7); Relatives + : mean of 636.6 cpm (SD f 234.7); Relatives - : mean of 543.3 cpm (SD f 373.3); Controls: mean of 43 1.7 cpm (SD* 134.5). 95% Confidence interval (CI) of the mean of the control group (whole blood and PBL 5 days): continuous line: CI for PBL 3 days: dashed line.
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of the Neurological
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Fig. 2. IL-2 Exogenous stimulation: (A) Whole blood: Patients: mean of 622.3 cpm (SD + 568.4); Relatives + : mean of 53 1.5 cpm (SD + 273.1); Relatives - : mean of 390.1 cpm (SD f 198.5); Controls: mean of 332.4 cpm (SD+ 125.7). (B) l PBL 3 days: Patients: mean of 989.2 cpm(SD+559.3);Relatives + : mean of 728.9 cpm (SD & 278.8); Relatives - : mean of 683.8 cpm (SD + 208.1); Controls: mean of 606.9 cpm(SDf 234.1).0 PBL 5 days:Patients: mean of 2257.6 cpm (SD + 1832.9); Relatives + : mean of 1669.8 cpm (SD + 906.7); Relatives - : mean of 1385.1 cpm(SDf 1144.2);Controls:meanof 863.8 cnm (SD f 644.9). 95% Confidence interval of the mean of the control group (whole blood and PBL 5 days): continuous line; CL for PBL 3 days: dashed iine.
demonstrate a statistical difference between HTLV-I myelopathy patients and the other groups. 3.2. Exogenous stimulation The response to IL-2 exogenous stimulation using whole blood showed a significant difference between patients suffering from HTLV-1 myelopathy and the control group (p < 0.05), but no difference was observed between seropositive and negative relatives (Fig. 2A). In PBL after 3 days of incubation a significant difference was obtained between patients with HTLV- 1 myelopathy and the control group ( p < 0.01) and seronegative relatives ( p < O.OS), although no difference was observed between seropositive and negative relatives. After 5 days of incubation in PBL stimulated with exogenous IL-2 no statistical difference was observed between the four groups (Fig. 2B). The lymphocyte proliferation with PHA stimulation did not show any statistical difference between the four groups in both systems, whole blood (patients: mean of 2331 cpm; relatives +: mean of 2749.7 cpm; relatives -: mean of 2126.7 cpm and control: mean of 2210.4) and PBL 3 days (patients: mean of 4508.4; relatives +: mean of 4988.5; relatives - : mean of 4918.5 and control: mean of 4594.7).
Fig. 3. Whole blood: (A) 0: spontaneous proliferation: mean of 225.5 (SDk94.7); 0: with Cs A: mean of 123.3 (SD+33.8). (B) 0: IL-2 exogenous stimulation: mean of 459 (SDk266); 0: with Cs A: mean of 214.1 (SD* 140.7).
M.J. Andrada-Serpa et al./ Journal of the Neurological
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Sciences 139 (1996) 7-14
Table 1 Mean percentage of inhibition by cyclosporin A
Spontaneous IL-2
Whole blood
PBL
40.7% 50.8%
43.3% 56.6%
myelopathies non related to HTLV-I infection (whole blood proliferation, patients: spontaneous: mean of 421.7 (SD ?c 319.41, IL-2: mean of 622.3 (SD f 555.9); other myelopathies: spontaneous: mean of 163.7 (SD + 35.1), IL-2: mean of 256.8 (SD f 84.9)). 3.3. Cyclosporin A inhibition
I
I
Fig. 4. PBL 3 days: (A) 0, spontaneous proloferation: mean 1105 (SD+ 596.9); 0. with Cs A: mean 508.5 (SD f Il. 1). (B) l ,IL-2 exogenous stimulation: mean 3076 (SD* 1793); 0, with Cs A: mean 963.8 (SD f 246.2).
Finally, a significant difference was observed in spontaneous proliferation ( p < 0.01) and IL-2 exogenous stimulation ( p < 0.05) when comparing patients with HTLV-1 myelopathy to a group of patients suffering from other
The addition of Cs A to the spontaneous proliferation induced a significant inhibition (p < 0.01) in both assays, PBL and whole blood. The addition of exogenous IL-2 did not abrogate the inhibition induced by Cs A (Figs. 3 and 4). Table 1 summarizes the results obtained, showing the mean percentage of inhibition by CsA in PBL and whole blood. 3.4. Patients, antibody titers and cytokines Table 2 shows the distribution of HTLV-I myelopathy patients according to sex and age as well as the serum
Table 2 Sex, age, serum antibody titer, level of TNF-a and IL-6, clinical disability and time of disease of 24 patients Patient
2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Sex/Age (years)
Antibody titer
IL-6 h/ml)
Time
C9lPl
TNF-(Y 6x/m])
DSS
MT-2
M/40 M/37 F/63 M/33 F/64 F/32 M/57 F/52 F/16 M/53 M/47 F/62 M/40 M/46 F/45 F/38 M/43 M/53 F/55 M/43 M/63 F/34 F/62 F/68
> 1:2,560 I :640 > 1:1,280 I:320 I:320 1:320 I:160 I : 1,280 I:320 1:320 1:320 I :2,560 1:160 I : 1,280 I :2,560 I :320 1:640 1:640 1:320 I :640 1:640 I :2,560 ]:I,280 1:1,280
> 1:2,560 I:],280 > 1:2,560 1:1,280 1:1,280 1:640 1:1,280 > I :2,560 1:320 1:1,280 I:],280 1:2,560 I:640 1: 1,280 I :2,560 I : 1,280 I : 1,280 1:640 1:320 ]:I,280 1:640 > 1:2,560 I:],280 1:1,280
0 0 0 0 0 0 8 0 0 11 ND 5 0 0 5 ND ND ND 0 0 0 ND 0 0
6 0 7 5.5 6 0 15 0 0 0 ND 5.5 0 0 5.5 0 ND ND 0 3 2 ND 0 6
4 4 2 6 6 7 5 6 6 2 5 7 3 3 3
108 32 5 16 84 19 60 68 36 120 72 384 19 4 36 -
M: male; F: female: ND: not done; DSS: disability status scale; time (month).
140 60 118 36 84 6 180
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antibody titer obtained by indirect immunofluorescence using MT-2 and C91pl cell lines and the results of IL-6 and TNF-cx levels in CSF. The healthy infected individuals were formed by family members, where 6 out of 9 were female spouses, 2 were offspring of patients and one a mother. The titers obtained with C91pl were higher than the ones with MT-2 and a significant difference was observed between titers of patients with HTLV-I myelopathies and healthy infected individuals. ((MT-2): patients: mean: 0.00211, SD + 0.00167; healthy: mean: 0.00885, SD * 0.00979, p = 0.003; (C91pl): patients: mean: 0.00100, SD k 0.00075; healthy: mean: 0.00842, SD & 0.01013, p = 0.0001). TNF-cx was detected only in four out of nineteen CSF samples, values ranging from 0 to 1 I pg/ml. IL-6 was detected in ten out of twenty CSF patients’ (range from 0 to 15 pg/ml). The disability status scale (Kurtzke, 1965) and time of disease are found in the table 2. No correlation was found between the time of disease, motor and sensory disability and the presence of cytokines in the CSF, or serum antibody titers.
4. Discussion Using two different systems to evaluate lymphocyte proliferation, PBL and whole blood, it was possible to demonstrate that Brazilian suffering from HTLV-I myelopathies present a high spontaneous lymphocyte proliferation in vitro. However, contrary to what has been published before (Itoyama et al., 1988b; Kramer et al., 1990) no statistical difference between seronegative and seropositive healthy relatives was found. The presence of exogenous IL-2 significantly increased patients’ lymphocyte proliferation in whole blood and in PBL 3 days when compared to the control group and seronegative relatives. The increased response to exogenous IL-2 probably represents the presence of activated T cells expressing IL-2 (Y receptor in the infected individuals. However, over the time, this difference seemed to disappear and no significant augmentation was observed in response to IL-2 in PBL cultures after 5 days. When PHA was used no difference was detected in the proliferative response between infected and non-infected individuals. Furthermore, in disagreement to what has been described in Japanese patients (Itoyama et al., 1988b), we were not able to demonstrate in Brazilian patients with HTLV-I myelopathies a decreased response to PHA stimulation. The mechanism whereby HTLV-I causes continuous activation of the immune system may result from multiple factors. Several studies have shown that the regulatory protein Tax transactivates multiple cellular genes involved in the T-cell activation, such as the gene for o-chain of the IL-2 receptor and the IL-2 gene, via the activation of the transcription factor NF-KB (Siekevitz et al., 1987; Tschachler et al., 1989). However, Hijllsberg et al. (1992)
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were unable to demonstrate the expression of IL-2 mRNA or IL- 2 protein in naturally infected T-cell clones thereby invalidating the possibility that their proliferation resulted solely from autocrine stimulation. The same authors showed that these HTLV-I infected T-cell clones were able to stimulate the proliferation of resting non-infected T-cells by a mechanism that involved the CD2/LFA-3 pathway (Wucherpfennig et al., 1992). The increased response to exogenous IL-2 observed in patients’ cells suggests that the expression of the IL-2 receptor may play a role in the continuous activation of the immune system and that this response may be due to the activation of non-infected T-cells by the CD2/LFA-3 pathway. This alternative activation pathway could explain the discrepant results described recently with Cs A, where naturally infected clones are resistant to Cs A and FK 506 suppression (Hollsberg et al., 1992) whereas, not cloned cell cultures were partially susceptible, as seen in our results (Natazuka et al., 1993). In the present work, Cs A inhibited spontaneous proliferation by nearly 40%, and this effect was not reversed by the presence of exogenous IL-2. These results suggest that Cs A might be an option in treating patients with HTLV-I myelopathies. The estimated number of infected leukocytes is around 10% (Richardson et al., 1990) and, although, Cs A might not inhibit HTLVI-infected T-cells, it could reduce the activation of bystander T-cells, which might play a main role in the pathogenesis of the disease. There are a number of evidences suggesting the importance of inflammation and immunological events during the course of the disease, and Cs A could be used especially for patients that did not attain a good response to high dose of steroids therapy (Osame et al., 1987) as this would decrease the immunologic hyperactivity observed. Another aspect that deserves consideration is the fact that according to Macatonia et al. (1992) HTLV-I may infect dendritic cells and the lack of these cells in proliferative assays abrogates the in vitro spontaneous lymphocyte proliferation. Dendritic cells are also susceptible to Cs A, as antigen presentation by these cells can be inhibited by the drug (Knight et al., 1988). On the other hand, dendritic cells represent less than 1% of peripheral blood cells and it is unknown if this amount is too small to stimulate proliferation in the whole blood assay. The presence of cytokines in the CSF was among the immunological abnormalities observed; IL-6 activity was detected in the CSF of nearly 50% of the patients and TNF-ok in 2 I %. These cytokines mediate natural immunity and have pleotropic activities in vivo and in vitro. In vitro production of IL-6 and TNF-cx have been described in the supematant of proliferating lymphocytes from of HTLVI/II infected individuals (La1 and Rudolph, 1991). We could not demonstrate a correlation between the presence of either cytokine, or their levels and the severity of the disease, although this has been previously suggested (Ohbo et al., 1991). The real meaning of the presence of IL-6 in
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the CSF remains unknown, Ohbo et al. suggested that steroid treatment decreases the level of IL-6 in the CSF and this immunological mediator may function as a marker for the activity of the disease. Finally, the presence of infected cells or the persistence of HTLV-I pX gene within the central nervous system may be relevant to the inflammatory response of HTLV-I myelopathy (Jacobson et al., 1992; Navarro-Roman et al., 1994). Similar to what has been observed in other endemic regions, our HTLV-I myelopathy patients present high antibody titers in the serum when compared to healthy infected individuals. It has been suggested that this enhanced antibody response to HTLV-I is the result of the increased virus load observed in these patients in comparison with healthy carriers (Shinzato et al., 1993). Our results show that HTLV-I myelopathy Brazilian patients present the same immunological abnormalities that have been described in patients from other geographic regions. The whole blood proliferation assay reflects the results obtained with separated peripheral blood cells and this assay may represent a good parameter for following therapeutic trials of HTLV-I myelopathy patients as it is economic and of simple execution. Our in vitro results with Cs A suggest that despite the lack of action on cloned infected T-cells, this drug may be a therapeutic option for treatment of this myelopathy as it significantly decreased the hyperimmune activity observed.
Acknowledgements Supported by grants from the Rio de Janeiro State Research Foundation (FAPERJ E- 11,’ 150.866/92), Brazilian Ministry of Health and World Health Organization (PAHO/WHO Ref. DRC/63/4.6).
References Andrada-Serpa, M.J., Dobbin, J.; Games, P.; Linhares, D.; Azevedo, J.G.; Hendriks, J.; Clayden, S.A.; Rumjanek, V.M.; Tedder, R.S. (1988) Incidence of retroviruses in some Brazilian groups. Immunol. Lett. 18: 15-18. Andrada-Serpa, M.J., Tosswill, J., Schor, D., Linhares, D., Dobbin, J., Pereira, M.S. (1989) Seroepidemiologic survey for antibodies to human retroviruses in human and non-human primates in Brazil. Int. J. Cancer 44: 389-393. Andrada-Serpa, M.J., Araujo, A.Q.C., Schor, D., Godoy, M., Afonso, C.R., Leite. A.C.B., Dultra, S., Nascimento, 0. (1993) Seroepidemiological study of HTLV-I/II infection among patients with myelopathy of unknown origin and their relatives in Rio de Janeiro. Can. J. Neurol. Sup1 4: S64. Andrada-Serpa, M.J.; Aratijo, A.Q.C.; Taffarel, M.; Schor, D.; Scheiner, M.A.M.; Ferreira, 0.; Schatzmayer. H.G. (1995) Detection and isolation of human T-cell leukemia/lymphoma virus type I (HTLV-I) from cultured lymphocytes of a Brazilian TSP/HAM patient, Braz. J. Med. Biol. Res. 28: 51-57. Araujo. A.Q-C., Ali, A., Newell, A., Dalgleish, A.G., Rudge, P. (1992)
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HTLV-I infection and neurological disease in Rio de Janeiro. J. Neurol. Neurosurg. Psychiatry 55: 153-155. Araujo, A.Q-C., Andrada-Serpa, M.J., Paulo-Filho, T.A., Rodrigues, M.T., Prado, L.A.F. (1995) Folliculitis decalvans and HTLV-I associated myelopathy/tropical spastic paraparesis. Clin. Infect. Dis. 20: 696-699. Blattner, W.A., Kalyanaraman, V.S., Robert-Guroff, M., Lister, D.A., Galton, D.A., Sarin. P.S., Crawford, M.H., Catvsky, D., Greaves, M., Gallo, R.C..(1982) The human type C retrovirus, HTLV, in blacks from the Caribbean region, and relationship to adult T-cell leukemia/lymphoma. Int. J. Cancer 30: 257-264. Chen, Y.A., Lee, T.H., Wiktor, S.Z., Shaw, G.M., Murphy, E.L., Blattrier, W.A., Essex, M. (1990). Type-specific antigens for serologic discrimination of HTLV-I and HTLV-II infection. Lancet 336: 11531155. Cortes, E., Detels, R., Aboulafia, D., Li, L.X., Moudgil, T., Alam, M., Bonecker, C., Gonzaga, A., Oyafuso, L., Tondo, M., Boite, C., Hammershlak, N.. Capitani, C., Slamon. D.J., Ho, D.D. (1989) HIV-I, HIV-2, and HTLV-I infection in high-risk groups in Brazil. N. Engl. J. Med. 320: 953-958. Gessain, A.. Barin, F., Vemant, J.C., Gout, O., Maurs, L., Calender, A.. DeThe, G. (1985) Antibodies to human T-lymphotrophic virus type I in patients with tropical spastic paraparesis. Lancet 2: 407-410. Hollsberg, P., Wucherpfennig. W.K., Ausubel. L.J., Calve, V., Bierer, B.E., Hafler, D.A. (1992) Characterization of HTLV-I in vivo infected T cell clones, IL-‘-Independent growth of nontransformed T cells. J. Immunol. 148: 3256-3263. Itoyama, Y., Minato, S., Kira, J., Goto. I., Sato. H.. Okochi, K.. Yamamoto, N. (1988a) Altered subsets of peripheral blood lymphocytes in patients with HTLV-I associated myelopathy (HAM). Neurology 38: 816-818. Itoyama, Y., Minato, S., Kira, J., Goto, I., Sato. H., Okochi, K.. Yamamoto. N. (1988b) Spontaneous proliferation of peripheral blood lymphocytes increased in patients with HTLV-I associated myelopathy. Neurology 38: 1302- 1307. Jacobson, S., Shida, H., McFdrlin, D.E., Fauci, A.S., Koenig, S. (1990) Circulating CD8 cytotoxic T lymphocytes specific for HTLV-I pX in patients with HTLV-I associated neurological disease. Nature 348: 245-248. Jacobson, S.. McFarlin, D.E., Robinson, S., Voskuhl, R., Martin. R., Brewah, A., Newell, A.M., Koenig, S. (1992) HTLV-I-specific cytotoxic T lymphocytes in the cerebrospinal fluid of patients with HTLV-I associated neurological disease. Ann. Neurol. 32: 65 l-657. Knight. S.C., Roberts, M., Macatonia, SE., Edwards. A.J. (1988) Cyclosporine blocks acquisition and presentation of antigen by dendritic cells. Studies with fluorescein isothiocyanate. Transplantation 46: 48-53. Kramer, A., Jacobson, S., Reuben, J.S., Murphy, E.L., Wiktor, S.Z., Cranston, B., Figuerod. J.P., Hanchard B., McFarlin, D., Blattner, W.A. (1990) Spontaneous lymphocyte proliferation is elevated in asymptomatic HTLV-I positive Jamaicans. In: W.A. Blattner (Ed.), Human Retrovirology: HTLV, Raven Press, New York, pp. 79-85. Kurtzke, J.F., (1965) Further notes on disability evaluation in multiple sclerosis with scale modification. Neurology 15: 654-661, LaGrenade, L., Hanchard, B., Fletcher, V., Cranston, B., Blattner, W. (1990) Infective dermatitis of Jamaican children: a marker for HTLV-I infection. Lancet 336: 1345- 1347. Lal, R.B. and Rudolph, D.L. (1991) Constitutive production of Interleukin-6 and Tumor Necrosis Factor-a from spontaneously proliferating T cells in patients with human T-cell lymphotropic virus type I. Blood 78: 57 l-574. Lal, R.B., Brodine, S.K.. Coligan, J.E.. Roberts, C.R. (1991) Differential antibody responsiveness to p19 gag results in serologiacal discrimination between human T lymphotropic virus type I and type 11. J. of Med. Viral. 35: 232-236. Macatonia, S.E., Cruickshank, J.K., Rudge, P., Knight. S.C. (1992) Dendritic cells from patients with tropical spastic paraparesis are
14
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et al. /Journal
of the Neurological
infected with HTLV-I and stimulate autologous lymphocytes proliferation. AIDS Hum. Res. Retrov. 8: 1699-1706. Mochizuki, M., Watanabe, T., Yamaguchi, K., Takatsuki, K., Yoshimura, K., Shirao, M., Nakashima, S., Mori, S., Araki, S., Miyata, N. (1992) HTLV-I Uveitis: A distinct clinical entity caused by HTLV-I. Jpn. J. Cancer Res. 83: 236-239. Moraes, V.L.G., Olej, B., De La Rocque, L., Rumjanek. V.M. (1989) Lack of sensitivity to ouabain in natural killer activity, FASEB J. 3: 2425-2429. Nagata, K., Ohtani, K., Nakamura, M., Sugamura, K. (1989) Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I encoded p40taX protein in the human t-cell line, Jurkat. J. Virol. 63: 3220-3226. Natazuka, T., Umemiya-Okada, T., Matsui, T., Saida, T., Nakao, Y. (1993) FK 506 and cyclosporin A regulate proliferation and protooncogene expression in HTLV-I-associated myelopathy/tropicalspastic-paraparesis-derived T cells, Int. J. Cancer 54: 348-354. Navarro-RomHn, L., Corbin, D.O.C., Katz, D., Callender, D.P.E., Prussia, P.R., Garriques, S., Fraser, H.S., Jaffe, E.S., Romlan, C.C. (1994) Human T-lymphotropic virus type I DNA in spinal cord of tropical spastic paraparesis with concomitant human T-lymphotropic virus type-1 negative Hodgkin’s disease. Human Pathol. 25: 1IO1 -1106. Ohbo, K., Sugamura, K., Sekizawa, T., Kogure, K. (1991) Interleukin-6 in cerebrospinal fluid of HTLV-I-associated myelopathy. Neurology 41: 594-595. Oliveira, M.S., Matutes, E., Famadas, L.C., Schulz, T.F., Calabro. M.L.. Nucci, M., Andrada-Serpa, M.J., Tedder, R.S., Weiss, R.A., Catovsky, D. (1990) Adult T-cell leukaemia/lymphoma in Brazil and its relation to HTLV-I. Lancet 336: 987-890. Osame. M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M.. Tara, M. (1986) HTLV-I associated myelopathy, a new clinical entity. Lancet 1: 1031-1032. Osame, M.. Matsumoto, M., Usuku, K., Izumo, O., Ijichi, N., Amitani, H., Tara. M., Igata, A. (1987) Chronic progressive myelopathy assocated with elevated antibodies to human T-lymphotropic virus type I
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and adult T-cell leukemia like cells. Ann. Neural. 21: 117-122. Osame, M. (1990) Review of WHO Kagoshima meeting and diagnostic guidelines for HAM/TSP. In: W.A. Blattner (Ed.), Human Retrovirology: HTLV, Raven Press, New York, pp. 191-197. Poiesz, B.J., Ruscetti, F.W., C&radar, A.F., Bunn, P.A., Minna, J.D., Gallo, R.C. (1980) Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of patients with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA 77: 7415-7419. Richardson, J.H., Edwards, A.J., Cruickshank, J.K., Rudge, P., Dalgleish, A. (1990) In vivo cellular tropism of human T-cell leukemia virus type I.J. Virol. 64: 5682-5687. Siekevitz, M., Feinberg, M.B., Holbrook. N., Wong-Staal, F., Greene, W.C. (1987) Activation of interleukin 2 and interleukin 2 receptor (Tat) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus type I. Proc. Nat]. Acad. Sci. USA 84: 5389-5393. Shinzato. 0.. Kamihira, S., Ikeda, S., Kondo, H., Kanda, T., Nagata, Y., Nakayama, E., Shiku, H. (1993) Relationship between the antiHTLV- 1 antibody level, the number of abnormal lymphocytes and the viral-genoma dose in HTLV-l-infected individuals. Int. J. Cancer 54: 208-212. Tschachler, E., Robert-Guroff, M., Gallo, R.C., Reitz, M.S. (1989) Human T-lymphotropic virus I infected T cells constitutively express lymphotoxin in viva. Blood 73: 194-201. Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K., Uchimo, H. (1977) Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood 50: 48 l-492. Vernant, J.C., Bellance, R., Buisson, G.G., Havard, S., Mikol, J. and Roman, G. (1990). Peripheral neuropathies and myositis associated to HTLV-I infection in Martinique. In: W.A. Blattner (Ed.), Human Retrovirology: HTLV, Raven Press, New York, pp. 225-235. Wucherpfennig, K.W., Hollsberg, P., Richardson, J.H., Benjamin, D., Hafler, D.A. (1992) T-cell activation by autologous human T-cell leukemia virus type I-infected T-cell clones. Proc. Natl. Acad. Sci. USA 89: 21 lo-21 14.
OF THE
NEUROLOGICAL SCIENCES
EL-SEVIER
Journal of the Neurological Sciences139(1996) 7- 14
Immunological features of HTLV-I myelopathy in Rio de Janeiro, Brazil, and in vitro effects of Cyclosporin A Maria J. Andrada-Serpa a** , Doris Schor a, Abelardo Q.-C. Aratijo b, Vivian M. Rumjanek ’ a Institute ’ Institute
de Biojkica
National de Cancer. Centro de Pesquisa Ba’sica, Praqa Cruz Vertnelha, 23, 20230-130, Rio de Janeiro, Brazil b Hospital Euandro Chagas, Funda@o Oswald0 Cm z, 21040-360, Rio de Janeiro, Brazil Caries Chagas Filho, Centro de Cikkias da Saiide, Uniuersidade Federal do Rio de Janeiro, Ilha do Fundiio. Rio de Janeiro, Brazil
Received 23 May 1995;revised 24 November 1995;accepted9 December 1995
Abstract Brazilian patients with HTLV-I myelopathy present a significant spontaneous lymphocyte proliferation (SLP), and an increased response to IL-2 exogenous stimulation, in both peripheral blood lymphocytes and in whole blood proliferative assays, when compared to the control group. High antibody titers against HTLV-I antigens were also observed in comparison to healthy seropositive individuals. IL-6 was detected in cerebrospinal fluid (CSF) of 50% of the patients (10 out of 20) and TNF-(Y in four out of nineteen individuals. No correlation was found between the presence of levels of cytokines IL-6 and TNF-a and duration or severity of disease. The addition of cyclosporin A (CsA) significantly inhibited SLP suggesting that this therapeutic agent should be studied in HTLV-1 myelopathy. Brazilian patients with HTLV-I myelopathy present the same immunological abnormalities described in other endemic regions. The whole blood assay reflects the same results of separated blood cells and, due to its rapid execution may be used as an assay to follow clinical trials. Keywords:
HTLV-I myelopathy; Healthy carriers: Spontaneouslymphocytic proliferation; Cyclosporin A inhibition; Interleukin-6; TNF-a
1. Introduction The human T lymphotropic virus type I (HTLV-I) is an etiologic agent of adult T-cell leukemia (ATL) (Uchiyama et al., 1977; Poiesz et al., 1980; Blattner et al., 1982) and is also associated with a chronic and progressive myelopa-
thy named tropical spastic paraparesis (TSP) or HTLV-Iassociated myelopathy (HAM) (Gessain et al., 1985; Osame et al., 1986). Recently, other diseases were also related to HTLV-I infection (Mochizuki et al., 1992; Vernant et al., 1990; LaGrenade et al., 1990; Tsukasaki et al., 1994; AraGjo et al., 1995). HTLV-1 myelopathy is clinically characterized by a progressive spastic paraparesis with antibodies to HTLV-I in the serum and cerebrospinal fluid (CSF). Although
* Corresponding author. Tel: +55 Fax: + 55 21 252-9598.
21 292-4110, extension
376/377/318.
0022-510X/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOO22-5 10X(96)00025- 1
HTLV-I has been isolated from CSF lymphocytes, the way through which the virus is related to the pathogenesis of this myelopathy still remains unknown. Several immunological abnormalities have been reported in these patients: (a) a hyperimmune activity with high antibody titers to HTLV-I in serum and cerebrospinal fluid (Osame et al., 19871, (b) the presence of activated lymphocytes in peripheral blood (Itoyama et al., 1988a), (c) the presence of spontaneous lymphocyte proliferation in vitro (Itoyama et al., 1988b; Jacobson et al., 1988) in patients and infected healthy individuals, and (d) a high level of cytotoxic T lymphocytes for HTLV-1 in the peripheral blood and in the cerebrospinal fluid (Jacobson et al., 1990; Jacobson et al., 1992). Furthermore, HTLV-I tax protein transactivates several cytokines and proto-oncogens genes involved in the activation and proliferation of lymphocytes (Siekevitz et al., 1987; Nagata et al., 1989; Tschachler et al., 19891, and a clinical improvement was observed, in some geographic regions with steroids therapy. All this indirect evidence suggests that inflammatory and immunological
8
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of the Neurological Sciences 139 (1996) 7-14
events may play a main role in the pathogenesis of the demyelinating process observed in these patients. Several reports have shown that Brazil is an endemic area for HTLV-I (Andrada-Serpa et al., 1988; AndradaSerpa et al., 1989; Cortes et al., 1989) and cases of ATL as well as HTLV-I myelopathy have already been described (Oliveira et al., 1990; Araujo et al., 1992; Andrada-Serpa et al., 1993; Andrada-Serpa et al., 1995). In the present study, the spontaneous and induced lymphocyte proliferation in Brazilian patients with HTLV- 1 myelopathy and HTLV-I infected healthy individuals was studied, as well as assessing the effect of cyclosporin A (CsA) in such cultures. Immunological activation at the central nervous system was also addressed and interleukin-6 (IL-6) and tumor necrosis alpha factor (TNF-o) quantified in the cerebrospinal fluid. The HTLV-I antibody titer in the patient’s sera was also measured.
2. Materials
and methods
2.1. Patients and serological tests Blood samples were obtained from the following groups: (a) twenty-four patients with HTLV-1 myelopathy; (b) nine HTLV-I seropositive family members; (c) forty-one HTLV-I seronegative family members; (d) five patients with myelopathy HTLV-I negative; and (e) nineteen healthy HTLV-I negative individuals as a control group. Patients had been attending the unit of HTLV neuropathies at the Hospital Evandro Chagas, FIOCRUZ, Rio de Janeiro. The diagnosis of HTLV-I myelopathy was based on the criteria established at the World Health Organization Kagoshima Meeting (Osame, 1990). Sera were screened by indirect immunofluorescence using two infected cell lines MT-2 and C91p1, the ones found positive were confirmed by a commercial western blot p21 enhanced (Cambridge Biotech Corporation, Worcester), the criteria of positivity was the expression of gag and env genes. All the sera from both the patients and the healthy carriers presented a blot pattern (~19 stronger than ~24) compatible with HTLV-I infection (Chen et al., 1990; La1 et al., 1991). The presence of HTLV-I antibody in the CSF patients as well as antibodies titers was performed by indirect immunofluorescence using both infected cell lines. 2.2. Immunological studies 2.2.1. Lymphocyte proliferation Peripheral blood lymphocytes (PBL) were separated from heparinized venous blood by density gradient centrifugation over Ficoll-Hypaque and washed twice in phosphate buffered saline (PBS). One hundred ~1 of the cell suspension (1 X lo6 cells/ml) in RPM1 1640 (Sigma
Company, Saint Louis, USA) supplemented with 2 mM L-glutamine, 100 U/ml of penicillin, 100 pg/ml of streptomycin and 10% of fetal calf serum (FCS) were plated in sterile flat-bottom 96-well microplates (Falcon 3072, Becton Dickinson, Oxnard, CA) and incubated with or without 5 kg/ml of PHA (Sigma Company, Saint Louis, USA), or with or without 10% of IL-2 (Lymphocult-T-LF, Biotest Diagnostics, Germany). Cyclosporin A (Sandoz, Basel, Switzerland) at a concentration of 0.5 or 1 pg/ml as well as exogenous stimulation (PHA and IL-2) were added at the beginning of the cultures. Cultures were maintained at 37°C in an atmosphere of 5% of CO, for three and five days. All cultures were performed in triplicate.
2.2.2. Whole blood proliferation Lymphocyte proliferation using whole blood (Moraes et al., 1989) was performed with blood diluted 5 times in RPM1 1640 without FCS and 100 pl were plated in triplicate with 100 pl of RPM1 1640 supplemented with 2 mM I-glutamine, 100 U/ml of penicillin, 100 p.g/ml of streptomycin and without FCS and incubated with or without 5 pi/ml of PHA or 10% of IL-2. Cyclosporin A at a concentration of 0.5 or 1 kg/ml was added at the beginning of the cultures. The cultures were maintained at 37°C in an atmosphere of 5% of CO, for five days. Lymphocyte proliferation was assessedby the uptake of [3H] thymidine. Cells were pulsed with 0.5 I.&i per well of [ 3H] thymidine (specific activity of 2 Ci/mmol) (Amersham International, Amersham, England, UK) for 6 h, harvested, and the radioactivity counted per minute (cpm) on a liquid scintillation counter (Beckman Instruments, Inc., CA, USA).
2.2.3. Cytokines Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-CY) were quantified in twenty CSF of patients with HTLV- 1 myelopathy. A commercial sandwich enzyme immunoassay (Research and Diagnostics Systems, Minneapolis, MN, USA) was used. Briefly, samples and standards were incubated in anti-IL-6 or anti-TNF-ol monoclonal antibody-coated polystyrene microtitre wells. Cytokine binds to antibody on the coated well, the unbound samples were removed by washing. After washing, an enzyme-linked polyclonal antibody specific for IL-6 or TNF-o was added to the wells to ‘sandwich’ the immobilized cytokine. Following a wash, a substrate solution was added to the wells and color develops in proportion to the amount of cytokine bound in the initial step. The reaction was stopped by the addition of 2N H,SO, and optical density was measured at 450 nm. According to the manufacturer’s instructions these immunoassays are able to detect TNF-o at a concentration greater than 4.8 pg/ml and IL-6 at one greater than 0.35 pg/ml. Standard curves
M.J. Andrada-Serpa et al. /Journal
were obtained with serial dilution of recombinant human TNF-a and of recombinant human IL-6 provided with immunoassays. 2.3. Statistical analysis Spontaneous proliferation and proliferation induced by exogenous stimulus were analyzed by Mann-Whitney Utest. The results obtained with cyclosporin A were evaluated by Wilcoxon’s test for paired data. Student’s t-test was also used to compare serological titers between patients and healthy infected individuals.
3. Results 3. I. Spontaneous lymphocyte proliferation In the whole blood system without exogenous stimulation a significant difference was observed between HTLV- 1 myelopathy patients and the other groups varying from
p < 0.01, when compared to the control group and seronegative relatives, to p < 0.05 when compared to seropositives. On the other hand, no difference was observed between seropositive and negative relatives (Fig. 1A). The results of spontaneous lymphocyte proliferation of PBL after 3 and 5 days of incubation are shown in the Fig. 1B. Patients with HTLV-1 myelopathy presented a significant spontaneous proliferation when compared to the control and seronegative groups (varying from p < 0.01 to p < 0.05). After 3 days of incubation a significant difference ( p < 0.05) was obtained between the patient group and seropositive individuals, but after 5 days of incubation this effect disappeared. No difference was observed between seropositive and negative relatives after 3 and 5 days of incubation. The levels of spontaneous proliferation obtained in our assays were lower than the ones obtained by other authors mainly due to the time interval used for pulse of [ ‘Hlthymidine (6 as opposed to 18 h), and perhaps to the specific activity of [3H]thymidine used in this study. Even with these experimental differences we were able to
H
0
0
l
9
of the Neurological Sciences 139 (19961 7-14
i .
3
.
TSP/HAM
Relatives+
Relatives-
Control
TSP/HM
Relatives+
Relatives-
Control
Fig. 1. Spontaneous proliferation: (A) Whole blood: Patients: mean of 421.7 cpm (SD k 3 19.4); Relatives + : mean of 243.2 cpm (SD + 159.9); Relatives - : mean of 206 cpm (SD k 8 1.5); Controls: mean of 175.2 cpm (SD + 53.3). (B) 0 PBL 3 days: Patients: mean of 702.5 cpm (SD + 289.9); Relatives + : mean of 458.8 cpm (SD f 115.5); Relatives - : mean of 503.3 cpm (SD* 129.1); Controls: mean of 506.9 cpm (SD* 162.4). 0 PBL 5 days: Patients: mean of 1225.6 cpm (SD + 608.7); Relatives + : mean of 636.6 cpm (SD f 234.7); Relatives - : mean of 543.3 cpm (SD f 373.3); Controls: mean of 43 1.7 cpm (SD* 134.5). 95% Confidence interval (CI) of the mean of the control group (whole blood and PBL 5 days): continuous line: CI for PBL 3 days: dashed line.
10
M.J. Andrada-Serpa et al. /Journal
I
\I
of the Neurological
Sciences 139 (1996) 7-14
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Fig. 2. IL-2 Exogenous stimulation: (A) Whole blood: Patients: mean of 622.3 cpm (SD + 568.4); Relatives + : mean of 53 1.5 cpm (SD + 273.1); Relatives - : mean of 390.1 cpm (SD f 198.5); Controls: mean of 332.4 cpm (SD+ 125.7). (B) l PBL 3 days: Patients: mean of 989.2 cpm(SD+559.3);Relatives + : mean of 728.9 cpm (SD & 278.8); Relatives - : mean of 683.8 cpm (SD + 208.1); Controls: mean of 606.9 cpm(SDf 234.1).0 PBL 5 days:Patients: mean of 2257.6 cpm (SD + 1832.9); Relatives + : mean of 1669.8 cpm (SD + 906.7); Relatives - : mean of 1385.1 cpm(SDf 1144.2);Controls:meanof 863.8 cnm (SD f 644.9). 95% Confidence interval of the mean of the control group (whole blood and PBL 5 days): continuous line; CL for PBL 3 days: dashed iine.
demonstrate a statistical difference between HTLV-I myelopathy patients and the other groups. 3.2. Exogenous stimulation The response to IL-2 exogenous stimulation using whole blood showed a significant difference between patients suffering from HTLV-1 myelopathy and the control group (p < 0.05), but no difference was observed between seropositive and negative relatives (Fig. 2A). In PBL after 3 days of incubation a significant difference was obtained between patients with HTLV- 1 myelopathy and the control group ( p < 0.01) and seronegative relatives ( p < O.OS), although no difference was observed between seropositive and negative relatives. After 5 days of incubation in PBL stimulated with exogenous IL-2 no statistical difference was observed between the four groups (Fig. 2B). The lymphocyte proliferation with PHA stimulation did not show any statistical difference between the four groups in both systems, whole blood (patients: mean of 2331 cpm; relatives +: mean of 2749.7 cpm; relatives -: mean of 2126.7 cpm and control: mean of 2210.4) and PBL 3 days (patients: mean of 4508.4; relatives +: mean of 4988.5; relatives - : mean of 4918.5 and control: mean of 4594.7).
Fig. 3. Whole blood: (A) 0: spontaneous proliferation: mean of 225.5 (SDk94.7); 0: with Cs A: mean of 123.3 (SD+33.8). (B) 0: IL-2 exogenous stimulation: mean of 459 (SDk266); 0: with Cs A: mean of 214.1 (SD* 140.7).
M.J. Andrada-Serpa et al./ Journal of the Neurological
11
Sciences 139 (1996) 7-14
Table 1 Mean percentage of inhibition by cyclosporin A
Spontaneous IL-2
Whole blood
PBL
40.7% 50.8%
43.3% 56.6%
myelopathies non related to HTLV-I infection (whole blood proliferation, patients: spontaneous: mean of 421.7 (SD ?c 319.41, IL-2: mean of 622.3 (SD f 555.9); other myelopathies: spontaneous: mean of 163.7 (SD + 35.1), IL-2: mean of 256.8 (SD f 84.9)). 3.3. Cyclosporin A inhibition
I
I
Fig. 4. PBL 3 days: (A) 0, spontaneous proloferation: mean 1105 (SD+ 596.9); 0. with Cs A: mean 508.5 (SD f Il. 1). (B) l ,IL-2 exogenous stimulation: mean 3076 (SD* 1793); 0, with Cs A: mean 963.8 (SD f 246.2).
Finally, a significant difference was observed in spontaneous proliferation ( p < 0.01) and IL-2 exogenous stimulation ( p < 0.05) when comparing patients with HTLV-1 myelopathy to a group of patients suffering from other
The addition of Cs A to the spontaneous proliferation induced a significant inhibition (p < 0.01) in both assays, PBL and whole blood. The addition of exogenous IL-2 did not abrogate the inhibition induced by Cs A (Figs. 3 and 4). Table 1 summarizes the results obtained, showing the mean percentage of inhibition by CsA in PBL and whole blood. 3.4. Patients, antibody titers and cytokines Table 2 shows the distribution of HTLV-I myelopathy patients according to sex and age as well as the serum
Table 2 Sex, age, serum antibody titer, level of TNF-a and IL-6, clinical disability and time of disease of 24 patients Patient
2 3 4 5 6 7 8 9 IO 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Sex/Age (years)
Antibody titer
IL-6 h/ml)
Time
C9lPl
TNF-(Y 6x/m])
DSS
MT-2
M/40 M/37 F/63 M/33 F/64 F/32 M/57 F/52 F/16 M/53 M/47 F/62 M/40 M/46 F/45 F/38 M/43 M/53 F/55 M/43 M/63 F/34 F/62 F/68
> 1:2,560 I :640 > 1:1,280 I:320 I:320 1:320 I:160 I : 1,280 I:320 1:320 1:320 I :2,560 1:160 I : 1,280 I :2,560 I :320 1:640 1:640 1:320 I :640 1:640 I :2,560 ]:I,280 1:1,280
> 1:2,560 I:],280 > 1:2,560 1:1,280 1:1,280 1:640 1:1,280 > I :2,560 1:320 1:1,280 I:],280 1:2,560 I:640 1: 1,280 I :2,560 I : 1,280 I : 1,280 1:640 1:320 ]:I,280 1:640 > 1:2,560 I:],280 1:1,280
0 0 0 0 0 0 8 0 0 11 ND 5 0 0 5 ND ND ND 0 0 0 ND 0 0
6 0 7 5.5 6 0 15 0 0 0 ND 5.5 0 0 5.5 0 ND ND 0 3 2 ND 0 6
4 4 2 6 6 7 5 6 6 2 5 7 3 3 3
108 32 5 16 84 19 60 68 36 120 72 384 19 4 36 -
M: male; F: female: ND: not done; DSS: disability status scale; time (month).
140 60 118 36 84 6 180
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antibody titer obtained by indirect immunofluorescence using MT-2 and C91pl cell lines and the results of IL-6 and TNF-cx levels in CSF. The healthy infected individuals were formed by family members, where 6 out of 9 were female spouses, 2 were offspring of patients and one a mother. The titers obtained with C91pl were higher than the ones with MT-2 and a significant difference was observed between titers of patients with HTLV-I myelopathies and healthy infected individuals. ((MT-2): patients: mean: 0.00211, SD + 0.00167; healthy: mean: 0.00885, SD * 0.00979, p = 0.003; (C91pl): patients: mean: 0.00100, SD k 0.00075; healthy: mean: 0.00842, SD & 0.01013, p = 0.0001). TNF-cx was detected only in four out of nineteen CSF samples, values ranging from 0 to 1 I pg/ml. IL-6 was detected in ten out of twenty CSF patients’ (range from 0 to 15 pg/ml). The disability status scale (Kurtzke, 1965) and time of disease are found in the table 2. No correlation was found between the time of disease, motor and sensory disability and the presence of cytokines in the CSF, or serum antibody titers.
4. Discussion Using two different systems to evaluate lymphocyte proliferation, PBL and whole blood, it was possible to demonstrate that Brazilian suffering from HTLV-I myelopathies present a high spontaneous lymphocyte proliferation in vitro. However, contrary to what has been published before (Itoyama et al., 1988b; Kramer et al., 1990) no statistical difference between seronegative and seropositive healthy relatives was found. The presence of exogenous IL-2 significantly increased patients’ lymphocyte proliferation in whole blood and in PBL 3 days when compared to the control group and seronegative relatives. The increased response to exogenous IL-2 probably represents the presence of activated T cells expressing IL-2 (Y receptor in the infected individuals. However, over the time, this difference seemed to disappear and no significant augmentation was observed in response to IL-2 in PBL cultures after 5 days. When PHA was used no difference was detected in the proliferative response between infected and non-infected individuals. Furthermore, in disagreement to what has been described in Japanese patients (Itoyama et al., 1988b), we were not able to demonstrate in Brazilian patients with HTLV-I myelopathies a decreased response to PHA stimulation. The mechanism whereby HTLV-I causes continuous activation of the immune system may result from multiple factors. Several studies have shown that the regulatory protein Tax transactivates multiple cellular genes involved in the T-cell activation, such as the gene for o-chain of the IL-2 receptor and the IL-2 gene, via the activation of the transcription factor NF-KB (Siekevitz et al., 1987; Tschachler et al., 1989). However, Hijllsberg et al. (1992)
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were unable to demonstrate the expression of IL-2 mRNA or IL- 2 protein in naturally infected T-cell clones thereby invalidating the possibility that their proliferation resulted solely from autocrine stimulation. The same authors showed that these HTLV-I infected T-cell clones were able to stimulate the proliferation of resting non-infected T-cells by a mechanism that involved the CD2/LFA-3 pathway (Wucherpfennig et al., 1992). The increased response to exogenous IL-2 observed in patients’ cells suggests that the expression of the IL-2 receptor may play a role in the continuous activation of the immune system and that this response may be due to the activation of non-infected T-cells by the CD2/LFA-3 pathway. This alternative activation pathway could explain the discrepant results described recently with Cs A, where naturally infected clones are resistant to Cs A and FK 506 suppression (Hollsberg et al., 1992) whereas, not cloned cell cultures were partially susceptible, as seen in our results (Natazuka et al., 1993). In the present work, Cs A inhibited spontaneous proliferation by nearly 40%, and this effect was not reversed by the presence of exogenous IL-2. These results suggest that Cs A might be an option in treating patients with HTLV-I myelopathies. The estimated number of infected leukocytes is around 10% (Richardson et al., 1990) and, although, Cs A might not inhibit HTLVI-infected T-cells, it could reduce the activation of bystander T-cells, which might play a main role in the pathogenesis of the disease. There are a number of evidences suggesting the importance of inflammation and immunological events during the course of the disease, and Cs A could be used especially for patients that did not attain a good response to high dose of steroids therapy (Osame et al., 1987) as this would decrease the immunologic hyperactivity observed. Another aspect that deserves consideration is the fact that according to Macatonia et al. (1992) HTLV-I may infect dendritic cells and the lack of these cells in proliferative assays abrogates the in vitro spontaneous lymphocyte proliferation. Dendritic cells are also susceptible to Cs A, as antigen presentation by these cells can be inhibited by the drug (Knight et al., 1988). On the other hand, dendritic cells represent less than 1% of peripheral blood cells and it is unknown if this amount is too small to stimulate proliferation in the whole blood assay. The presence of cytokines in the CSF was among the immunological abnormalities observed; IL-6 activity was detected in the CSF of nearly 50% of the patients and TNF-ok in 2 I %. These cytokines mediate natural immunity and have pleotropic activities in vivo and in vitro. In vitro production of IL-6 and TNF-cx have been described in the supematant of proliferating lymphocytes from of HTLVI/II infected individuals (La1 and Rudolph, 1991). We could not demonstrate a correlation between the presence of either cytokine, or their levels and the severity of the disease, although this has been previously suggested (Ohbo et al., 1991). The real meaning of the presence of IL-6 in
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the CSF remains unknown, Ohbo et al. suggested that steroid treatment decreases the level of IL-6 in the CSF and this immunological mediator may function as a marker for the activity of the disease. Finally, the presence of infected cells or the persistence of HTLV-I pX gene within the central nervous system may be relevant to the inflammatory response of HTLV-I myelopathy (Jacobson et al., 1992; Navarro-Roman et al., 1994). Similar to what has been observed in other endemic regions, our HTLV-I myelopathy patients present high antibody titers in the serum when compared to healthy infected individuals. It has been suggested that this enhanced antibody response to HTLV-I is the result of the increased virus load observed in these patients in comparison with healthy carriers (Shinzato et al., 1993). Our results show that HTLV-I myelopathy Brazilian patients present the same immunological abnormalities that have been described in patients from other geographic regions. The whole blood proliferation assay reflects the results obtained with separated peripheral blood cells and this assay may represent a good parameter for following therapeutic trials of HTLV-I myelopathy patients as it is economic and of simple execution. Our in vitro results with Cs A suggest that despite the lack of action on cloned infected T-cells, this drug may be a therapeutic option for treatment of this myelopathy as it significantly decreased the hyperimmune activity observed.
Acknowledgements Supported by grants from the Rio de Janeiro State Research Foundation (FAPERJ E- 11,’ 150.866/92), Brazilian Ministry of Health and World Health Organization (PAHO/WHO Ref. DRC/63/4.6).
References Andrada-Serpa, M.J., Dobbin, J.; Games, P.; Linhares, D.; Azevedo, J.G.; Hendriks, J.; Clayden, S.A.; Rumjanek, V.M.; Tedder, R.S. (1988) Incidence of retroviruses in some Brazilian groups. Immunol. Lett. 18: 15-18. Andrada-Serpa, M.J., Tosswill, J., Schor, D., Linhares, D., Dobbin, J., Pereira, M.S. (1989) Seroepidemiologic survey for antibodies to human retroviruses in human and non-human primates in Brazil. Int. J. Cancer 44: 389-393. Andrada-Serpa, M.J., Araujo, A.Q.C., Schor, D., Godoy, M., Afonso, C.R., Leite. A.C.B., Dultra, S., Nascimento, 0. (1993) Seroepidemiological study of HTLV-I/II infection among patients with myelopathy of unknown origin and their relatives in Rio de Janeiro. Can. J. Neurol. Sup1 4: S64. Andrada-Serpa, M.J.; Aratijo, A.Q.C.; Taffarel, M.; Schor, D.; Scheiner, M.A.M.; Ferreira, 0.; Schatzmayer. H.G. (1995) Detection and isolation of human T-cell leukemia/lymphoma virus type I (HTLV-I) from cultured lymphocytes of a Brazilian TSP/HAM patient, Braz. J. Med. Biol. Res. 28: 51-57. Araujo. A.Q-C., Ali, A., Newell, A., Dalgleish, A.G., Rudge, P. (1992)
13
HTLV-I infection and neurological disease in Rio de Janeiro. J. Neurol. Neurosurg. Psychiatry 55: 153-155. Araujo, A.Q-C., Andrada-Serpa, M.J., Paulo-Filho, T.A., Rodrigues, M.T., Prado, L.A.F. (1995) Folliculitis decalvans and HTLV-I associated myelopathy/tropical spastic paraparesis. Clin. Infect. Dis. 20: 696-699. Blattner, W.A., Kalyanaraman, V.S., Robert-Guroff, M., Lister, D.A., Galton, D.A., Sarin. P.S., Crawford, M.H., Catvsky, D., Greaves, M., Gallo, R.C..(1982) The human type C retrovirus, HTLV, in blacks from the Caribbean region, and relationship to adult T-cell leukemia/lymphoma. Int. J. Cancer 30: 257-264. Chen, Y.A., Lee, T.H., Wiktor, S.Z., Shaw, G.M., Murphy, E.L., Blattrier, W.A., Essex, M. (1990). Type-specific antigens for serologic discrimination of HTLV-I and HTLV-II infection. Lancet 336: 11531155. Cortes, E., Detels, R., Aboulafia, D., Li, L.X., Moudgil, T., Alam, M., Bonecker, C., Gonzaga, A., Oyafuso, L., Tondo, M., Boite, C., Hammershlak, N.. Capitani, C., Slamon. D.J., Ho, D.D. (1989) HIV-I, HIV-2, and HTLV-I infection in high-risk groups in Brazil. N. Engl. J. Med. 320: 953-958. Gessain, A.. Barin, F., Vemant, J.C., Gout, O., Maurs, L., Calender, A.. DeThe, G. (1985) Antibodies to human T-lymphotrophic virus type I in patients with tropical spastic paraparesis. Lancet 2: 407-410. Hollsberg, P., Wucherpfennig. W.K., Ausubel. L.J., Calve, V., Bierer, B.E., Hafler, D.A. (1992) Characterization of HTLV-I in vivo infected T cell clones, IL-‘-Independent growth of nontransformed T cells. J. Immunol. 148: 3256-3263. Itoyama, Y., Minato, S., Kira, J., Goto. I., Sato. H.. Okochi, K.. Yamamoto, N. (1988a) Altered subsets of peripheral blood lymphocytes in patients with HTLV-I associated myelopathy (HAM). Neurology 38: 816-818. Itoyama, Y., Minato, S., Kira, J., Goto, I., Sato. H., Okochi, K.. Yamamoto. N. (1988b) Spontaneous proliferation of peripheral blood lymphocytes increased in patients with HTLV-I associated myelopathy. Neurology 38: 1302- 1307. Jacobson, S., Shida, H., McFdrlin, D.E., Fauci, A.S., Koenig, S. (1990) Circulating CD8 cytotoxic T lymphocytes specific for HTLV-I pX in patients with HTLV-I associated neurological disease. Nature 348: 245-248. Jacobson, S.. McFarlin, D.E., Robinson, S., Voskuhl, R., Martin. R., Brewah, A., Newell, A.M., Koenig, S. (1992) HTLV-I-specific cytotoxic T lymphocytes in the cerebrospinal fluid of patients with HTLV-I associated neurological disease. Ann. Neurol. 32: 65 l-657. Knight. S.C., Roberts, M., Macatonia, SE., Edwards. A.J. (1988) Cyclosporine blocks acquisition and presentation of antigen by dendritic cells. Studies with fluorescein isothiocyanate. Transplantation 46: 48-53. Kramer, A., Jacobson, S., Reuben, J.S., Murphy, E.L., Wiktor, S.Z., Cranston, B., Figuerod. J.P., Hanchard B., McFarlin, D., Blattner, W.A. (1990) Spontaneous lymphocyte proliferation is elevated in asymptomatic HTLV-I positive Jamaicans. In: W.A. Blattner (Ed.), Human Retrovirology: HTLV, Raven Press, New York, pp. 79-85. Kurtzke, J.F., (1965) Further notes on disability evaluation in multiple sclerosis with scale modification. Neurology 15: 654-661, LaGrenade, L., Hanchard, B., Fletcher, V., Cranston, B., Blattner, W. (1990) Infective dermatitis of Jamaican children: a marker for HTLV-I infection. Lancet 336: 1345- 1347. Lal, R.B. and Rudolph, D.L. (1991) Constitutive production of Interleukin-6 and Tumor Necrosis Factor-a from spontaneously proliferating T cells in patients with human T-cell lymphotropic virus type I. Blood 78: 57 l-574. Lal, R.B., Brodine, S.K.. Coligan, J.E.. Roberts, C.R. (1991) Differential antibody responsiveness to p19 gag results in serologiacal discrimination between human T lymphotropic virus type I and type 11. J. of Med. Viral. 35: 232-236. Macatonia, S.E., Cruickshank, J.K., Rudge, P., Knight. S.C. (1992) Dendritic cells from patients with tropical spastic paraparesis are
14
M.J. Andrada-Serpa
et al. /Journal
of the Neurological
infected with HTLV-I and stimulate autologous lymphocytes proliferation. AIDS Hum. Res. Retrov. 8: 1699-1706. Mochizuki, M., Watanabe, T., Yamaguchi, K., Takatsuki, K., Yoshimura, K., Shirao, M., Nakashima, S., Mori, S., Araki, S., Miyata, N. (1992) HTLV-I Uveitis: A distinct clinical entity caused by HTLV-I. Jpn. J. Cancer Res. 83: 236-239. Moraes, V.L.G., Olej, B., De La Rocque, L., Rumjanek. V.M. (1989) Lack of sensitivity to ouabain in natural killer activity, FASEB J. 3: 2425-2429. Nagata, K., Ohtani, K., Nakamura, M., Sugamura, K. (1989) Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I encoded p40taX protein in the human t-cell line, Jurkat. J. Virol. 63: 3220-3226. Natazuka, T., Umemiya-Okada, T., Matsui, T., Saida, T., Nakao, Y. (1993) FK 506 and cyclosporin A regulate proliferation and protooncogene expression in HTLV-I-associated myelopathy/tropicalspastic-paraparesis-derived T cells, Int. J. Cancer 54: 348-354. Navarro-RomHn, L., Corbin, D.O.C., Katz, D., Callender, D.P.E., Prussia, P.R., Garriques, S., Fraser, H.S., Jaffe, E.S., Romlan, C.C. (1994) Human T-lymphotropic virus type I DNA in spinal cord of tropical spastic paraparesis with concomitant human T-lymphotropic virus type-1 negative Hodgkin’s disease. Human Pathol. 25: 1IO1 -1106. Ohbo, K., Sugamura, K., Sekizawa, T., Kogure, K. (1991) Interleukin-6 in cerebrospinal fluid of HTLV-I-associated myelopathy. Neurology 41: 594-595. Oliveira, M.S., Matutes, E., Famadas, L.C., Schulz, T.F., Calabro. M.L.. Nucci, M., Andrada-Serpa, M.J., Tedder, R.S., Weiss, R.A., Catovsky, D. (1990) Adult T-cell leukaemia/lymphoma in Brazil and its relation to HTLV-I. Lancet 336: 987-890. Osame. M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M.. Tara, M. (1986) HTLV-I associated myelopathy, a new clinical entity. Lancet 1: 1031-1032. Osame, M.. Matsumoto, M., Usuku, K., Izumo, O., Ijichi, N., Amitani, H., Tara. M., Igata, A. (1987) Chronic progressive myelopathy assocated with elevated antibodies to human T-lymphotropic virus type I
Sciences 139 (1996)
7-14
and adult T-cell leukemia like cells. Ann. Neural. 21: 117-122. Osame, M. (1990) Review of WHO Kagoshima meeting and diagnostic guidelines for HAM/TSP. In: W.A. Blattner (Ed.), Human Retrovirology: HTLV, Raven Press, New York, pp. 191-197. Poiesz, B.J., Ruscetti, F.W., C&radar, A.F., Bunn, P.A., Minna, J.D., Gallo, R.C. (1980) Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of patients with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA 77: 7415-7419. Richardson, J.H., Edwards, A.J., Cruickshank, J.K., Rudge, P., Dalgleish, A. (1990) In vivo cellular tropism of human T-cell leukemia virus type I.J. Virol. 64: 5682-5687. Siekevitz, M., Feinberg, M.B., Holbrook. N., Wong-Staal, F., Greene, W.C. (1987) Activation of interleukin 2 and interleukin 2 receptor (Tat) promoter expression by the trans-activator (tat) gene product of human T-cell leukemia virus type I. Proc. Nat]. Acad. Sci. USA 84: 5389-5393. Shinzato. 0.. Kamihira, S., Ikeda, S., Kondo, H., Kanda, T., Nagata, Y., Nakayama, E., Shiku, H. (1993) Relationship between the antiHTLV- 1 antibody level, the number of abnormal lymphocytes and the viral-genoma dose in HTLV-l-infected individuals. Int. J. Cancer 54: 208-212. Tschachler, E., Robert-Guroff, M., Gallo, R.C., Reitz, M.S. (1989) Human T-lymphotropic virus I infected T cells constitutively express lymphotoxin in viva. Blood 73: 194-201. Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K., Uchimo, H. (1977) Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood 50: 48 l-492. Vernant, J.C., Bellance, R., Buisson, G.G., Havard, S., Mikol, J. and Roman, G. (1990). Peripheral neuropathies and myositis associated to HTLV-I infection in Martinique. In: W.A. Blattner (Ed.), Human Retrovirology: HTLV, Raven Press, New York, pp. 225-235. Wucherpfennig, K.W., Hollsberg, P., Richardson, J.H., Benjamin, D., Hafler, D.A. (1992) T-cell activation by autologous human T-cell leukemia virus type I-infected T-cell clones. Proc. Natl. Acad. Sci. USA 89: 21 lo-21 14.