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Immunological alterations in uncomplicated Plasmodium falciparum malaria. Relationship between parasitaemia and indicators of macrophage activation
Acta Tropica, 46(1989)351-359 Elsevier
351
ACTROP 00042
Immunological alterations in uncomplicated Plasmodium f alciparum malaria. Relationship between parasitaemia and indicators of macrophage activation P.G. Kremsner 1, H. Feldmeier 1, G.M. Zotter x, R. Jansen-Rosseck 1, W. Graninger 2, R.M. R o c h a 3 a n d U. Bienzle 1 1Landesinstitut fiir Tropenmedizin, Berlin, Germany, 2Universitiitsklinik fiir Chemotherapie, University of Vienna, Austria, and 3Superintendencia de Campanhas de Saude Publica Acre, Brazil
(Received 1 February 1989; accepted 3 May 1989) Numerical alterations of circulating lymphocytes were investigated in 37 Brazilian patients with uncomplicated Plasmodium falciparurn malaria and in a group of 15 healthy controls. The number of CD4 + T helper/inducer cells was significantly lower in patients than controls, whereas absolute numbers of CD8 + suppressor/cytotoxic T cells did not differ between the groups. TNF and neopterin levels were markedly increased in the plasma of patients and remained slightly elevated after chemotherapy with clindamycin. Neopterin, but not TNF levels, were signficantly correlated with parasitaemia. TNF was inversely related to monocyte counts. Interferon 7 could not be detected in the plasma of control subjects and was observed in only one patient. We conclude that in uncomplicated falciparum malaria the distribution of phenotypes of circulating lymphocytes are altered slightly and that the high plasma levelsof TNF and neopterin indicate excessive release of these molecules by activated macrophages and the activation of cellular immune mechanisms during the infection. Key words: Tumour necrosis factor; Neopterin; Lymphocyte phenotypes; Falciparum malaria
Introduction A l t h o u g h in recent years considerable progress has been achieved in the field o f m a l a r i a i m m u n o l o g y , the m e c h a n i s m s which protect i n h a b i t a n t s o f e n d e m i c areas from severe infection are still a m a t t e r o f debate ( M c G r e g o r , 1987). After intensive investigations o f a n t i b o d y - m e d i a t e d i m m u n i t y to merozoite a n d sporozoite antigens, interest has n o w focused o n cell-mediated i m m u n e responses. I m m u n o r e g u l a t o r y mdlecules such as i n t e r f e r o n - g a m m a ( I F N - 7 ) a n d t u m o u r necrosis factor ( T N F ) m a y play a n i m p o r t a n t role in i m m u n i t y , as they could help or activate B cells a n d m a c r o p h a g e s ( O c k e n h o u s e et al., 1984) or directly interfere with parasite growth (Clark, 1987; Schofield et al., 1987). However, as far as T N F is concerned, this cytokine m a y also be involved in the p a t h o l o g y of the infection (Clark, 1987). I n addition, there is Correspondence address." Dr. P.G. Kremsner, Landesinstitut f/Jr Tropenmedizin, K6nigin-Elisabeth-Stral3e 32, D-1000 Berlin 19, Germany.
0001-706X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)
352 evidence that acute malaria can lead to depression of the cellular immune response (Tosta, 1987), as measured in vitro and by in vivo assays of delayed type hypersensitivity (Wyler and Oppenheim, 1974; Tosta et al., 1983; Troye-Blomberg et al., 1985; Kremsner et al., in preparation), and a reduced activity of natural killer cells (Stach et al:, 1986). This is accompanied by a decrease of CD4 lymphocytes (Stach et al., 1986). However, it is not known whether activation of T cells and macrophages is related to parasitaemia and clinical expression of the disease. Therefore we decided to investigate the occurrence ofphenotypic alterations of circulating lymphocytes and the presence of circulating immunoregulatory molecules in a group of patients with uncomplicated Plasmodium falciparum infection and control subjects.
Subjects and Methods
Location of study: the study took place in an outpatient clinic in Rio Branco, Acre, Brazil, in the Amazonian rain forest, where multiresistant P.falciparum occurs with a high frequency (Kremsner et al., 1989a). Patients and controls: all patients admitted to the study met the following criteria: (1) Older than 15 years. (2) Monoinfection with P.falciparum. (3) No intake of antimalarial drugs during the preceding week. (4) No clinical complications (cerebral malaria etc.). (5) No pregnancy. Twenty-eight patients were male, nine were female; they ranged in age from 16 to 50 years (median 26 years). Fifteen healthy age- and sex-matched subjects from Rio Branco served as controls, none of them had parasitaemia. Informed consent was obtained. Clinical examinations: physical examinations were performed on admission between 8.00 and 11.00 a.m. Axillary temperature was measured. Daily clinical checks were performed during the first week. Chemotherapy: because of the high frequency of multiresistant isolates in study area and the proven efficacy of clindamycin in these cases (Kremsner et al., 1989b) patients received clindamycin orally in a dosage of 5 mg/kg twice a day for 5 consecutive days. The control subjects took the same regimen. Laboratory examinations: a heparinized venous blood sample was drawn on admission and after 5 days of clindamycin therapy. Plasma was stored at - 20°C for 2-4 months until tested. Haematocrit and the differential white blood cell counts were performed by routine methods. Daily parasite counts were performed in thick blood smears as described elsewhere (Kremsner et al., 1989b). Peripheral blood mononuclear cells (PMNC) were isolated from heparinized blood by density gradient centrifugation (LeucoPREP, Becton Dickinson, CA, U.S.A.). Mouse mofioclonal antibodies against CD3, CD4 and CD8 (Behring, Vienna, Austria) were employed to determine lymphocyte subpopulations as described previously (Gastl et al., 1984). Fluoresceinated affinity-purified goat anti-mouse IgG was used as a second reagent. One-hundred lymphocytes were scored under a fluorescence microscope with epiluminescence. Plasma TNF, neopterin and IFN-~ concentrations were measured by radio immunoassays (RIA), following the manufacturers' instructions (Medgenix, Fleurus, Belgium; Henning, Berlin, Germany). The detection limits for T N F and IFN are 100 pg/ml and 2 U/ml, respectively. TNF values of less than 100 pg/ml were scored as
353 0 pg/ml. The normal mean serum neopterin value of healthy Europeans is 5.9_+ 1.8 pmol/ml. Statistics: comparisons between patients and controls were calculated by the Mann-Whitney test, and between parameters by the Spearman rank test. Differences between T N F and neopterin values on admission and 5 days later were determined by the Wilcoxon rank sum test. The median and its 95% confidence intervals (95% C.I.) were used as descriptive measures.
Results
Clinical and parasitological findings All patients suffered from a mild to moderate course of their disease. The symptoms are listed in Table 1. Axillary temperature ranged from 36.0 to 39.1°C (median 37.0°C). As expected haematocrit values were significantly decreased in the patients: median 38% as compared to 45% in the controls. Eighty-four percent of the patients and only one of the controls had experienced at least one malaria attack during the preceding 12 months. During treatment with clindamycin the patients clinical condition improved rapidly. Most of the patients were free of symptoms on the 3rd or 4th day after initiation of chemotherapy. In three of the 37 patients slight headache and nausea persisted until the 5th day. Upon admission parasite density varied considerably: range 12-32680 parasites/~tl (Table 1). Five days later parasitaemia was absent in 21 patients. In 16 patients thick blood films remained positive. However, parasitaemia had decreased (range 0-255 asexual parasites/~tl) in these patients. By day 7 all patients were parasite-free.
Lymphocyte phenotypes The number of P M N C and the lymphocyte subsets in patients and controls are shown in Table 2. The counts of lymphocytes and monocytes were similar in patients TABLE 1 Clinical and parasitologicalfindingsin the 37 patients on admission n Splenomegaly Headache Nausea Backache Hepatomegaly Fever ( > 37.5°C) Vomiting Vertigo Diarrhoea Chills Asexual parasites/l~l
% 23 19 18 10 9 6 6 3 2 1
median 2220
62 51 49 27 24 16 16 8 5 3 95% confidenceinterval (1530-4572)
354 TABLE 2 Peripheral mononuclear blood cells in patients and controls on admission
Monocytes Lymphocytes CD3+ cells CD4+ cells CD8 + cells
Patients (n = 37)
Controls (n = 15)
Median
95% C.I?
Median
95% C.I."
1l0 1650 1159 614 469
(80 130) ( 1400 2200) (942 1404) (497 781) (422-560)
114 2000 1327 891 454
(70-144) ( 1500 2500) (1073 1674) (710 1167) (302-538)
Significance of difference
n.s. b
n.s. n.s. p=0.003 n.s.
Data indicate blood cells per gl of blood. " 95% confidence interval. b n.s. = not significant. a n d controls. C D 3 + cells were slightly, t h o u g h n o t significantly, decreased. H o w ever, in the patients the n u m b e r o f C D 4 + h e l p e r / i n d u c e r T cells was significantly diminished, whereas C D 8 + s u p p r e s s o r / c y t o t o x i c T cells seemed to be slightly elevated in relative n u m b e r s . T h e d i m i n i s h e d n u m b e r o f C D 4 + T cells is clearly d e m o n s t r a t e d by the C D 4 + / C D 8 + ratio: m e d i a n 1.3 in patients versus 2.0 in c o n t r o l s ( p < 0.05).
Immunoregulatory molecules The p l a s m a c o n c e n t r a t i o n s o f T N F a n d n e o p t e r i n are s u m m a r i z e d in T a b l e 3. T N F was detected in the p l a s m a o f 21 ( 5 7 % ) p a t i e n t s ( m e d i a n 130 pg/ml, 9 5 % C.I. 0 to 190 pg/ml), but in n o n e o f 15 c o n t r o l s ( p _< 0.001). T h e m e d i a n p l a s m a level o f n e o p t e r i n was a l m o s t 7 times higher in p a t i e n t s t h a n in c o n t r o l s (38.2 p m o l / m l versus 6.0 p m o l / m l , p < 0.001) a l t h o u g h n e o p t e r i n c o n c e n t r a t i o n in the latter did not differ f r o m t h a t o f h e a l t h y E u r o p e a n s . In patients a positive c o r r e l a t i o n was o b s e r v e d between i n d i v i d u a l c o n c e n t r a t i o n s o f T N F a n d n e o p t e r i n ( r h o = 0 . 3 0 , p < 0 . 0 5 ) . Surprisingly, p l a s m a n e o p t e r i n was higher in p a t i e n t s with a high p a r a s i t a e m i a ( > 5000 asexual p a r a s i t e s / g l ) than in those with low p a r a s i t a e m i a ( < 1000 asexual parasites/gl, p < 0.1, Fig. 1). M o r e o v e r , regression analysis between p a r a s i t e density a n d n e o p t e r i n c o n c e n t r a t i o n after c l i n d a m y c i n t r e a t m e n t showed a weak, but TABLE 3 Plasma concentrations of TNF and neopterin in patients and controls on admission Patients Median TNF (pg/ml) Neopterin (pmol/ml)
130 38.2
(n =
37)
Controls (n
95% C.I."
Median
- 15)
Significance of difference
95% C.l?
(0 190)
0b
(0-0)
p< 0.001
(27.9-44.0)
6.0
(5.8 8.2)
p < 0.001
a95% Confidence interval. h0 for TNF means lower than 100 pg/ml.
355
E 100 --
E o. "E
z
:
|
50-I M~
M~IM~
:
I < 1000
i
I 1001-5000
I > 5000 Parasites/jJI
Fig. 1. Neopterin plasma concentrations in three groups of patients with different levels of parasitaemia on admission. Horizontal bars indicate the median and 95% confidence intervals.
significant correlation (rho = 0.31, p < 0.05). Such a relationship was not observed between parasitaemia and the level of TNF. As TNF is preponderately released by cells of the monocyte/macrophage lineage, regression analysis was performed between the number of circulating monocytes and plasma TNF in individual patients. Interestingly, an inverse correlation was observed (rho = -0.31, p < 0.05). When correlation was calculated using only patients with detectable levels of TNF, the correlation did just not reach the level of significance
(p>0.05). IFN-v could not be detected in any plasma sample of the controls, and in only one of the 37 malaria patients.
Effect of chemotherapy The effect of chemotherapy with clindamycin on the plasma concentration of TNF and neopterin of 37 patients is depicted in Figs. 2 and 3. Five days after initiation of treatment neopterin values showed a slight, though not significant decrease (median on admission 38.2 pmoi/ml and 26.7 pmol/ml 5 days later, p<0.1, Fig. 2). However, TNF significantly decreased after clindamycin treatment (median < 100 pg/ml as compared to 130 pg/ml before treatment, p = 0.02, Fig. 3). In only 11 patients TNF concentration in plasma remained above 100 pg/ml.
Discussion
Lymphocytopenia is a common finding in falciparum malaria. Our data indicate that this is due to a reduced number of circulating T cells. Within the T cell compartment
356
200
~15C
E CL .C
ilJ
z 100
50 --$--
-!: -! -
-|--: -
I I I day 0
! I day 5
Fig. 2. Neopterin plasma concentration in 37 patients before and after clindamycin treatment (p = 0.07). Horizontal bars indicate the median and 95% confidence intervals.
I 20001
1000
2
E !
•
t:i-100
< 100
I
I
day 0
day 5
Fig. 3. T N F plasma concentration in 37 patients before and after clindamycin treatment (p = 0.02).
357
C D 4 + helper/inducer cells were diminished in absolute and relative numbers, leading to a median C D 4 + / C D 8 + ratio of 1.3. The selective decrease of C D 4 + T cells in our patients confirms previous observations in children and in P. falciparumor P. vivax-infected adults from different endemic areas (Stach et al., 1986; Merino et al., 1986). As under in vitro conditions the stimulation of PMNC of acutely ill patients with schizont-derived antigens induces the clonal expansion of C D 4 + cells (Pink et al., 1987; Sinigaglia et al., 1987; Chizzolini et al., 1988), the finding in vivo of a reduced number of circulating C D 4 + cells remains intriguing. It is consistent, though, with the clinically known state of transitional immunosuppression observed in P. falciparum malaria (Merino et al., 1986; Kremsner et al.; in preparation). The biological role of T N F is still a matter of discussion. There is evidence for an important role of T N F as a mediator of'endotoxic shock and cachexia (Beutler et al., 1985; Tracey et al., 1987), and recently this cytokine has also been implicated in malarial pathophysiology (Clark et al., 1987). In our patients elevated plasma concentrations of T N F were observed upon admission independently of the peripheral density of parasites. The detection of TNF in plasma implies a production in great excess and continuous turnover of this cytokine, as the biological plasma half life is approximately 6 min (Beutler et al., 1985). After 5 days ofclindamycin therapy T N F levels decreased markedly. On admission we found an inverse correlation between the concentration of TNF in plasma and the number of peripheral monocytes in patients. Monocytes/macrophages are actived during acute malaria, which is indicated by an increase of Fc receptor molecules on their surface (Ward et al., 1984). T N F as a free molecule will bind to specific receptors near the activation site, but will also gain access into blood circulation. Activated mononuclear phagocytes may be sequestered in peripheral capillary vessels at the activation site, where high concentrations of plasmodial antigen are found. This may lead to a diminution in the number of peripheral monocytes. This hypothesis is in agreement with findings that accumulations of mononuclear phagocytes are scattered in the vascular system throughout the body of malaria-infected mice (Franz et al., 1988). IFN- 7 activity was detected in sera of acutely ill patients with P. falciparum malaria (Rhode-Feuillette et al., 1985). However, in only one of our patients it could be detected by RIA. Until recently bioassays were used for the detection of IFN. As the specificity of RIA is higher, but sensitivity lower, this seems to be the most likely explanation for the differing results. We do not know whether IFN concentrations below our detection limit exert a substantial biological activity. Neopterin is a low molecular weight compound derived biosynthetically from guanosine triphosphate. The only known sources of neopterin are human and other primate monocyte/macrophages after activation with lymphokines such as IFN- 7 (Huber et al., 1984). The increase of neopterin in plasma of patients with uncomplicated falciparum malaria confirms previous observations in other malaria patients (Reibnegger et al., 1984). Moreover, we demonstrated a positive correlation between parasitaemia and circulating neopterin. This is in agreement with the assumption based on experimental findings in neoplastic diseases that neopterin release is coupled with early events of activated cell-mediated immunity (Huber et al., 1987) and that malaria parasites induce not only humoral but also cellular immune responses (McGregor, 1987). Elevated plasma levels of neopterin do not, however, indicate that
358 cells o f the i m m u n e system are f u n c t i o n i n g a d e q u a t e l y . I n c r e a s e d n e o p t e r i n has been o b s e r v e d in p a t i e n t s with viral diseases as well as in h e p a t o s p l e n i c schistosomiasis, a disease a s s o c i a t e d with i m p a i r e d c e l l u l a r - m e d i a t e d i m m u n i t y ( K e r n , 1985; Z w i n g e n berger et al., 1988). T h e persistence o f elevated p l a s m a c o n c e n t r a t i o n in s o m e b u t not all p a t i e n t s 5 d a y s after initiation o f c l i n d a m y c i n t r e a t m e n t is intriguing. H o w e v e r , n e o p t e r i n levels were f o u n d to be highest in p a t i e n t s with high p a r a s i t a e m i a . In view o f o u r o w n similar findings in p a t i e n t s with recrudescence o f P. vivax infection, it is t e m p t i n g to speculate t h a t persistent high levels o f n e o p t e r i n in c i r c u l a t i o n m a y indicate c o n t i n u e d a c t i v a t i o n o f c e l l - m e d i a t e d i m m u n i t y as a c o n s e q u e n c e o f c o n t i n u o u s presence o f m a l a r i a parasites.
Acknowledgements P . G . K . was on a fellowship at S U C A M Acre, Brazil, in 1987 (grant P 62 33 M, F F W F Austria). W e t h a n k Leitz A u s t r i a for p r o v i d i n g a m i c r o s c o p e . W e t h a n k Behring in A u s t r i a for p r o v i d i n g m o n o c l o n a l a n t i b o d i e s to l y m p h o c y t e subsets. W e t h a n k Becton D i c k i n s o n in A u s t r i a for p r o v i d i n g Leuco P R E P . W e t h a n k D a g m a r F e n g l e r - D o p p for excellent a n d skilfull l a b o r a t o r y assistance. W e t h a n k Dr. M a r i a Luisa a n d Dr. F r a n c i M e i r y for their help.
References Beutler, B.A., Milsark, I.W. and Cerami, A. (1985) Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. J. Immunol. 135, 3972-3977. Chizzolini, C. and Perrin, L. (1988) Antigen specific and MHC restricted Plasmodiumfalciparum-induced human T lymphocyte clones. J. Immunol. 137, 1022-1028. Clark, I.A. (1987) Monokines and lymphokines in malarial pathology. Ann. Trop. Med. Parasitol. 81, 577-585. Franz, D.R., Lim, D.S., Baze, W.B., Arimbalam, S., Lee, M. and Lewis G.E. (1988) Pathologic activity of Plasmodium berghei prevented but not reversed by dexamethasone. Am. J. Trop. Med. Hyg. 38, 249-254. Gastl, G.A., Feldmeier, H., Doehring, E., Kortmann, C., Daffalla, A.A. and Peter, H.H. (1984) Numerical and functional alterations of lymphocytes in human schistosomiasis. Scand. J. Immunol. 19, 469 479. Huber, C., Batchelor, J.R., Fuchs, D., Hausen, A., Lang, A., Niederwieser, D., Reibnegger, G., Swetley, P., Troppmair, I. and Wachter, H. (1984) Immune response associated production of neopterin. Release of macrophages primarily under control of interferon gamma. J. Exp. Med. 160, 310 316. Huber, C., Troppmair, J., Rokos, H. and Curtius, H.C. (1987) Neopterin heute. Dtsch. Med. Wochenschr. 112, 107-113. Kern, P. (1985) Erh6hte Serum Neopterin Werte bei verschiedenen Viruserkrankungen und bei AIDS. Mitt. Osterr. Ges. Tropenmed. Parasitol. 7, 19-22. Kremsner, P.G., Zotter, G.M., Feldmeier, H., Graninger, W., Kollaritsch, H., Wiedermann, G., Rocha, R.M. and Wernsdorfer, W.H. (1989a) In vitro drug sensitivity of Plasmodiumfah'iparum in Acre, Brazil. Bull. WHO, in press. Kremsner, P.G., Zotter, G.M., Feldmeier, H., Graninger, W., Westerman, R.L. and Rocha, R.M. (1989b) Clindamycin treatment of falciparum malaria in Brazil. J. Antimicrob. Chemother. 23, 275-281. McGregor, I.A. (1987) Malarial immunity: current trends and prospects. Ann. Trop. Med. Parasitol. 81, 647-656. Merino, F., Layrisse, Z., Godoy, G. and Volcan, G. (1986) Immunoregulatory alterations on Plasmodium falciparum and Plasmodium vivax infections. Trop. Med. Parasitol. 37, 241-244. Ockenhouse, C.F., Schulman, S. and Shear, H.L. (1984) Induction of crisis forms in the human parasite P.faciparum by interferon-activated monocyte derived macrophages. J. Immunol. 133, 1601 1608.
359 Pink, J.R.L., Rijnbeek, A., Reber-Liske, R. and Sinigaglia, F. (1987) Plasmodium falciparum specific human T cell clones: recognition of different parasite antigens. Eur. J. Immunol. 17, 193-196. Reibnegger, G., Boonpucknavig, V., Fuchs, D., Hausen, A., Schmutzhard, E. and Wachter, H. (1984) Urinary neopterin is elevated in patients with malaria. Trans. R. Trop. Med. Hyg. 78, 545-546. Rhode-Feuillette, A., Bellosguardo, M., Druilhe, P., Ballet, J.J., Chousterman, S., Carnivet, M. and Peries, J. (1985) The interferon compartment of the immune response in human malaria. II. Presence of serum interferon gamma following the acute attack. J. Interferon Res. 5, 169-178. Schofield, L., Villaquiran, J., Ferreira, A., Schellekens, H., Nussenzweig, R. and Nussenzweig, V. (1987) ~?Interferon, CD8 + T cells and antibodies required for immunity to malaria sporozoites. Nature 330, 664-666. Sinigaglia, F., Matile, H. and Pink, J.R.L. (1987) Plasmodiumfalciparum specific human T cell clones: evidence for helper and cytotoxic activities. Eur. J. Immunol. 17, 187-192. Stach, S.L., Ufrenoy, E., Roffi, J. and Bach, M.A. (1986) T cell subsets and natural killer activity in Plasmodiumfalciparum-infected children. Clin. Immunol. Immunopath01. 38, 129-134. Tosta, C.E. (1987) Immunodeficiency associated with malaria. Rev. Soc. Brasil. Med. Trop. 20, I-5. Tosta, C.E., Ruiz, G. and Wedderburn, N. (1983) Effects of lethal and nonlethal malaria on the mononuclear phagocyte system. Rev. Soc,. Brasil. Med. Trop. 16, 58-67. Tracey, K.J., Fang, Y., Hesse, D.G., Menogue, K.R., Lee, A.T., Kuo, G.C., Lowry, S.F. and Cerami, A. (1987) Anti cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330, 662-664. Troye-Blomberg, M., Andersson, G., Stoczkowska, M., Shabo, R., Romero, P., Patarroyo, E., Wigzell, H. and Perlmann, P. (1985) Production of IL 2 and IFN by T cells from malaria patients in response to Plasmodiurn falciparum or erythrocyte antigens in vitro. J. Immunol. 135, 3498-3504. Ward, K.N., Warrell, M.J., Rhodes, J., Looaresuwan, S. and White, N.J. (1984) Altered expression of human monocyte Fc receptors in Plasmodiumfalciparum malaria. Infect. Immun. 44, 623 626. Wyler, D.J. and Oppenheim, J.J. (1974) Lymphocyte transformation in human Plasrnodiumfalciparum malaria. J. lmmunol. 113, 449 454. Zwingenberger, K., Feldmeier, H., Nogueira Queiroz, J.A., Vergetti Siqueira, J.G., Farias Auto, H., Alencar, J.E. and Bienzle, U. (1988) Liver involvement in human schistosomiasis mansoni. Assessment by immunological and biochemical markers. Parasitol. Res. 74, 448-455.
351
ACTROP 00042
Immunological alterations in uncomplicated Plasmodium f alciparum malaria. Relationship between parasitaemia and indicators of macrophage activation P.G. Kremsner 1, H. Feldmeier 1, G.M. Zotter x, R. Jansen-Rosseck 1, W. Graninger 2, R.M. R o c h a 3 a n d U. Bienzle 1 1Landesinstitut fiir Tropenmedizin, Berlin, Germany, 2Universitiitsklinik fiir Chemotherapie, University of Vienna, Austria, and 3Superintendencia de Campanhas de Saude Publica Acre, Brazil
(Received 1 February 1989; accepted 3 May 1989) Numerical alterations of circulating lymphocytes were investigated in 37 Brazilian patients with uncomplicated Plasmodium falciparurn malaria and in a group of 15 healthy controls. The number of CD4 + T helper/inducer cells was significantly lower in patients than controls, whereas absolute numbers of CD8 + suppressor/cytotoxic T cells did not differ between the groups. TNF and neopterin levels were markedly increased in the plasma of patients and remained slightly elevated after chemotherapy with clindamycin. Neopterin, but not TNF levels, were signficantly correlated with parasitaemia. TNF was inversely related to monocyte counts. Interferon 7 could not be detected in the plasma of control subjects and was observed in only one patient. We conclude that in uncomplicated falciparum malaria the distribution of phenotypes of circulating lymphocytes are altered slightly and that the high plasma levelsof TNF and neopterin indicate excessive release of these molecules by activated macrophages and the activation of cellular immune mechanisms during the infection. Key words: Tumour necrosis factor; Neopterin; Lymphocyte phenotypes; Falciparum malaria
Introduction A l t h o u g h in recent years considerable progress has been achieved in the field o f m a l a r i a i m m u n o l o g y , the m e c h a n i s m s which protect i n h a b i t a n t s o f e n d e m i c areas from severe infection are still a m a t t e r o f debate ( M c G r e g o r , 1987). After intensive investigations o f a n t i b o d y - m e d i a t e d i m m u n i t y to merozoite a n d sporozoite antigens, interest has n o w focused o n cell-mediated i m m u n e responses. I m m u n o r e g u l a t o r y mdlecules such as i n t e r f e r o n - g a m m a ( I F N - 7 ) a n d t u m o u r necrosis factor ( T N F ) m a y play a n i m p o r t a n t role in i m m u n i t y , as they could help or activate B cells a n d m a c r o p h a g e s ( O c k e n h o u s e et al., 1984) or directly interfere with parasite growth (Clark, 1987; Schofield et al., 1987). However, as far as T N F is concerned, this cytokine m a y also be involved in the p a t h o l o g y of the infection (Clark, 1987). I n addition, there is Correspondence address." Dr. P.G. Kremsner, Landesinstitut f/Jr Tropenmedizin, K6nigin-Elisabeth-Stral3e 32, D-1000 Berlin 19, Germany.
0001-706X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)
352 evidence that acute malaria can lead to depression of the cellular immune response (Tosta, 1987), as measured in vitro and by in vivo assays of delayed type hypersensitivity (Wyler and Oppenheim, 1974; Tosta et al., 1983; Troye-Blomberg et al., 1985; Kremsner et al., in preparation), and a reduced activity of natural killer cells (Stach et al:, 1986). This is accompanied by a decrease of CD4 lymphocytes (Stach et al., 1986). However, it is not known whether activation of T cells and macrophages is related to parasitaemia and clinical expression of the disease. Therefore we decided to investigate the occurrence ofphenotypic alterations of circulating lymphocytes and the presence of circulating immunoregulatory molecules in a group of patients with uncomplicated Plasmodium falciparum infection and control subjects.
Subjects and Methods
Location of study: the study took place in an outpatient clinic in Rio Branco, Acre, Brazil, in the Amazonian rain forest, where multiresistant P.falciparum occurs with a high frequency (Kremsner et al., 1989a). Patients and controls: all patients admitted to the study met the following criteria: (1) Older than 15 years. (2) Monoinfection with P.falciparum. (3) No intake of antimalarial drugs during the preceding week. (4) No clinical complications (cerebral malaria etc.). (5) No pregnancy. Twenty-eight patients were male, nine were female; they ranged in age from 16 to 50 years (median 26 years). Fifteen healthy age- and sex-matched subjects from Rio Branco served as controls, none of them had parasitaemia. Informed consent was obtained. Clinical examinations: physical examinations were performed on admission between 8.00 and 11.00 a.m. Axillary temperature was measured. Daily clinical checks were performed during the first week. Chemotherapy: because of the high frequency of multiresistant isolates in study area and the proven efficacy of clindamycin in these cases (Kremsner et al., 1989b) patients received clindamycin orally in a dosage of 5 mg/kg twice a day for 5 consecutive days. The control subjects took the same regimen. Laboratory examinations: a heparinized venous blood sample was drawn on admission and after 5 days of clindamycin therapy. Plasma was stored at - 20°C for 2-4 months until tested. Haematocrit and the differential white blood cell counts were performed by routine methods. Daily parasite counts were performed in thick blood smears as described elsewhere (Kremsner et al., 1989b). Peripheral blood mononuclear cells (PMNC) were isolated from heparinized blood by density gradient centrifugation (LeucoPREP, Becton Dickinson, CA, U.S.A.). Mouse mofioclonal antibodies against CD3, CD4 and CD8 (Behring, Vienna, Austria) were employed to determine lymphocyte subpopulations as described previously (Gastl et al., 1984). Fluoresceinated affinity-purified goat anti-mouse IgG was used as a second reagent. One-hundred lymphocytes were scored under a fluorescence microscope with epiluminescence. Plasma TNF, neopterin and IFN-~ concentrations were measured by radio immunoassays (RIA), following the manufacturers' instructions (Medgenix, Fleurus, Belgium; Henning, Berlin, Germany). The detection limits for T N F and IFN are 100 pg/ml and 2 U/ml, respectively. TNF values of less than 100 pg/ml were scored as
353 0 pg/ml. The normal mean serum neopterin value of healthy Europeans is 5.9_+ 1.8 pmol/ml. Statistics: comparisons between patients and controls were calculated by the Mann-Whitney test, and between parameters by the Spearman rank test. Differences between T N F and neopterin values on admission and 5 days later were determined by the Wilcoxon rank sum test. The median and its 95% confidence intervals (95% C.I.) were used as descriptive measures.
Results
Clinical and parasitological findings All patients suffered from a mild to moderate course of their disease. The symptoms are listed in Table 1. Axillary temperature ranged from 36.0 to 39.1°C (median 37.0°C). As expected haematocrit values were significantly decreased in the patients: median 38% as compared to 45% in the controls. Eighty-four percent of the patients and only one of the controls had experienced at least one malaria attack during the preceding 12 months. During treatment with clindamycin the patients clinical condition improved rapidly. Most of the patients were free of symptoms on the 3rd or 4th day after initiation of chemotherapy. In three of the 37 patients slight headache and nausea persisted until the 5th day. Upon admission parasite density varied considerably: range 12-32680 parasites/~tl (Table 1). Five days later parasitaemia was absent in 21 patients. In 16 patients thick blood films remained positive. However, parasitaemia had decreased (range 0-255 asexual parasites/~tl) in these patients. By day 7 all patients were parasite-free.
Lymphocyte phenotypes The number of P M N C and the lymphocyte subsets in patients and controls are shown in Table 2. The counts of lymphocytes and monocytes were similar in patients TABLE 1 Clinical and parasitologicalfindingsin the 37 patients on admission n Splenomegaly Headache Nausea Backache Hepatomegaly Fever ( > 37.5°C) Vomiting Vertigo Diarrhoea Chills Asexual parasites/l~l
% 23 19 18 10 9 6 6 3 2 1
median 2220
62 51 49 27 24 16 16 8 5 3 95% confidenceinterval (1530-4572)
354 TABLE 2 Peripheral mononuclear blood cells in patients and controls on admission
Monocytes Lymphocytes CD3+ cells CD4+ cells CD8 + cells
Patients (n = 37)
Controls (n = 15)
Median
95% C.I?
Median
95% C.I."
1l0 1650 1159 614 469
(80 130) ( 1400 2200) (942 1404) (497 781) (422-560)
114 2000 1327 891 454
(70-144) ( 1500 2500) (1073 1674) (710 1167) (302-538)
Significance of difference
n.s. b
n.s. n.s. p=0.003 n.s.
Data indicate blood cells per gl of blood. " 95% confidence interval. b n.s. = not significant. a n d controls. C D 3 + cells were slightly, t h o u g h n o t significantly, decreased. H o w ever, in the patients the n u m b e r o f C D 4 + h e l p e r / i n d u c e r T cells was significantly diminished, whereas C D 8 + s u p p r e s s o r / c y t o t o x i c T cells seemed to be slightly elevated in relative n u m b e r s . T h e d i m i n i s h e d n u m b e r o f C D 4 + T cells is clearly d e m o n s t r a t e d by the C D 4 + / C D 8 + ratio: m e d i a n 1.3 in patients versus 2.0 in c o n t r o l s ( p < 0.05).
Immunoregulatory molecules The p l a s m a c o n c e n t r a t i o n s o f T N F a n d n e o p t e r i n are s u m m a r i z e d in T a b l e 3. T N F was detected in the p l a s m a o f 21 ( 5 7 % ) p a t i e n t s ( m e d i a n 130 pg/ml, 9 5 % C.I. 0 to 190 pg/ml), but in n o n e o f 15 c o n t r o l s ( p _< 0.001). T h e m e d i a n p l a s m a level o f n e o p t e r i n was a l m o s t 7 times higher in p a t i e n t s t h a n in c o n t r o l s (38.2 p m o l / m l versus 6.0 p m o l / m l , p < 0.001) a l t h o u g h n e o p t e r i n c o n c e n t r a t i o n in the latter did not differ f r o m t h a t o f h e a l t h y E u r o p e a n s . In patients a positive c o r r e l a t i o n was o b s e r v e d between i n d i v i d u a l c o n c e n t r a t i o n s o f T N F a n d n e o p t e r i n ( r h o = 0 . 3 0 , p < 0 . 0 5 ) . Surprisingly, p l a s m a n e o p t e r i n was higher in p a t i e n t s with a high p a r a s i t a e m i a ( > 5000 asexual p a r a s i t e s / g l ) than in those with low p a r a s i t a e m i a ( < 1000 asexual parasites/gl, p < 0.1, Fig. 1). M o r e o v e r , regression analysis between p a r a s i t e density a n d n e o p t e r i n c o n c e n t r a t i o n after c l i n d a m y c i n t r e a t m e n t showed a weak, but TABLE 3 Plasma concentrations of TNF and neopterin in patients and controls on admission Patients Median TNF (pg/ml) Neopterin (pmol/ml)
130 38.2
(n =
37)
Controls (n
95% C.I."
Median
- 15)
Significance of difference
95% C.l?
(0 190)
0b
(0-0)
p< 0.001
(27.9-44.0)
6.0
(5.8 8.2)
p < 0.001
a95% Confidence interval. h0 for TNF means lower than 100 pg/ml.
355
E 100 --
E o. "E
z
:
|
50-I M~
M~IM~
:
I < 1000
i
I 1001-5000
I > 5000 Parasites/jJI
Fig. 1. Neopterin plasma concentrations in three groups of patients with different levels of parasitaemia on admission. Horizontal bars indicate the median and 95% confidence intervals.
significant correlation (rho = 0.31, p < 0.05). Such a relationship was not observed between parasitaemia and the level of TNF. As TNF is preponderately released by cells of the monocyte/macrophage lineage, regression analysis was performed between the number of circulating monocytes and plasma TNF in individual patients. Interestingly, an inverse correlation was observed (rho = -0.31, p < 0.05). When correlation was calculated using only patients with detectable levels of TNF, the correlation did just not reach the level of significance
(p>0.05). IFN-v could not be detected in any plasma sample of the controls, and in only one of the 37 malaria patients.
Effect of chemotherapy The effect of chemotherapy with clindamycin on the plasma concentration of TNF and neopterin of 37 patients is depicted in Figs. 2 and 3. Five days after initiation of treatment neopterin values showed a slight, though not significant decrease (median on admission 38.2 pmoi/ml and 26.7 pmol/ml 5 days later, p<0.1, Fig. 2). However, TNF significantly decreased after clindamycin treatment (median < 100 pg/ml as compared to 130 pg/ml before treatment, p = 0.02, Fig. 3). In only 11 patients TNF concentration in plasma remained above 100 pg/ml.
Discussion
Lymphocytopenia is a common finding in falciparum malaria. Our data indicate that this is due to a reduced number of circulating T cells. Within the T cell compartment
356
200
~15C
E CL .C
ilJ
z 100
50 --$--
-!: -! -
-|--: -
I I I day 0
! I day 5
Fig. 2. Neopterin plasma concentration in 37 patients before and after clindamycin treatment (p = 0.07). Horizontal bars indicate the median and 95% confidence intervals.
I 20001
1000
2
E !
•
t:i-100
< 100
I
I
day 0
day 5
Fig. 3. T N F plasma concentration in 37 patients before and after clindamycin treatment (p = 0.02).
357
C D 4 + helper/inducer cells were diminished in absolute and relative numbers, leading to a median C D 4 + / C D 8 + ratio of 1.3. The selective decrease of C D 4 + T cells in our patients confirms previous observations in children and in P. falciparumor P. vivax-infected adults from different endemic areas (Stach et al., 1986; Merino et al., 1986). As under in vitro conditions the stimulation of PMNC of acutely ill patients with schizont-derived antigens induces the clonal expansion of C D 4 + cells (Pink et al., 1987; Sinigaglia et al., 1987; Chizzolini et al., 1988), the finding in vivo of a reduced number of circulating C D 4 + cells remains intriguing. It is consistent, though, with the clinically known state of transitional immunosuppression observed in P. falciparum malaria (Merino et al., 1986; Kremsner et al.; in preparation). The biological role of T N F is still a matter of discussion. There is evidence for an important role of T N F as a mediator of'endotoxic shock and cachexia (Beutler et al., 1985; Tracey et al., 1987), and recently this cytokine has also been implicated in malarial pathophysiology (Clark et al., 1987). In our patients elevated plasma concentrations of T N F were observed upon admission independently of the peripheral density of parasites. The detection of TNF in plasma implies a production in great excess and continuous turnover of this cytokine, as the biological plasma half life is approximately 6 min (Beutler et al., 1985). After 5 days ofclindamycin therapy T N F levels decreased markedly. On admission we found an inverse correlation between the concentration of TNF in plasma and the number of peripheral monocytes in patients. Monocytes/macrophages are actived during acute malaria, which is indicated by an increase of Fc receptor molecules on their surface (Ward et al., 1984). T N F as a free molecule will bind to specific receptors near the activation site, but will also gain access into blood circulation. Activated mononuclear phagocytes may be sequestered in peripheral capillary vessels at the activation site, where high concentrations of plasmodial antigen are found. This may lead to a diminution in the number of peripheral monocytes. This hypothesis is in agreement with findings that accumulations of mononuclear phagocytes are scattered in the vascular system throughout the body of malaria-infected mice (Franz et al., 1988). IFN- 7 activity was detected in sera of acutely ill patients with P. falciparum malaria (Rhode-Feuillette et al., 1985). However, in only one of our patients it could be detected by RIA. Until recently bioassays were used for the detection of IFN. As the specificity of RIA is higher, but sensitivity lower, this seems to be the most likely explanation for the differing results. We do not know whether IFN concentrations below our detection limit exert a substantial biological activity. Neopterin is a low molecular weight compound derived biosynthetically from guanosine triphosphate. The only known sources of neopterin are human and other primate monocyte/macrophages after activation with lymphokines such as IFN- 7 (Huber et al., 1984). The increase of neopterin in plasma of patients with uncomplicated falciparum malaria confirms previous observations in other malaria patients (Reibnegger et al., 1984). Moreover, we demonstrated a positive correlation between parasitaemia and circulating neopterin. This is in agreement with the assumption based on experimental findings in neoplastic diseases that neopterin release is coupled with early events of activated cell-mediated immunity (Huber et al., 1987) and that malaria parasites induce not only humoral but also cellular immune responses (McGregor, 1987). Elevated plasma levels of neopterin do not, however, indicate that
358 cells o f the i m m u n e system are f u n c t i o n i n g a d e q u a t e l y . I n c r e a s e d n e o p t e r i n has been o b s e r v e d in p a t i e n t s with viral diseases as well as in h e p a t o s p l e n i c schistosomiasis, a disease a s s o c i a t e d with i m p a i r e d c e l l u l a r - m e d i a t e d i m m u n i t y ( K e r n , 1985; Z w i n g e n berger et al., 1988). T h e persistence o f elevated p l a s m a c o n c e n t r a t i o n in s o m e b u t not all p a t i e n t s 5 d a y s after initiation o f c l i n d a m y c i n t r e a t m e n t is intriguing. H o w e v e r , n e o p t e r i n levels were f o u n d to be highest in p a t i e n t s with high p a r a s i t a e m i a . In view o f o u r o w n similar findings in p a t i e n t s with recrudescence o f P. vivax infection, it is t e m p t i n g to speculate t h a t persistent high levels o f n e o p t e r i n in c i r c u l a t i o n m a y indicate c o n t i n u e d a c t i v a t i o n o f c e l l - m e d i a t e d i m m u n i t y as a c o n s e q u e n c e o f c o n t i n u o u s presence o f m a l a r i a parasites.
Acknowledgements P . G . K . was on a fellowship at S U C A M Acre, Brazil, in 1987 (grant P 62 33 M, F F W F Austria). W e t h a n k Leitz A u s t r i a for p r o v i d i n g a m i c r o s c o p e . W e t h a n k Behring in A u s t r i a for p r o v i d i n g m o n o c l o n a l a n t i b o d i e s to l y m p h o c y t e subsets. W e t h a n k Becton D i c k i n s o n in A u s t r i a for p r o v i d i n g Leuco P R E P . W e t h a n k D a g m a r F e n g l e r - D o p p for excellent a n d skilfull l a b o r a t o r y assistance. W e t h a n k Dr. M a r i a Luisa a n d Dr. F r a n c i M e i r y for their help.
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