Acute P. falciparum malaria induces a loss of CD28 − T IFN-γ producing cells

Parasite Immunology, 2002, 24, 545– 548

Research note – RESEARCH Loss of CD28 NOTE T cellsLtd. in acute P. falciparum malaria Blackwell Publishing

Acute P. falciparum malaria induces a loss of CD28– T IFN-γ producing cells KÅRE KEMP1, BARTHOLOMEW D. AKANMORI2, JØRGEN A. L. KURTZHALS1,2,3, VICTORIA ADABAYERI3, BAMENLA Q. GOKA3 & LARS HVIID1 1

Centre for Medical Parasitology at Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet) and Institute for Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark, 2Immunology Unit, Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana, 3Department of Child Health, Korle-Bu Teaching Hospital, University of Ghana Medical School, Accra, Ghana

SUMMARY

RESEARCH NOTE

P. falciparum malaria is associated with increased activation among peripheral lymphocytes. In the present study, we investigated markers of susceptibility to apoptosis and expression of IFN-γ and IL-4 by CD28– and CD28+ T cells in West African children with acute P. falciparum malaria. The study showed increased susceptibility to apoptosis and cytokine production among T lymphocytes during acute malaria but also that T cells, in particular IFN-γ producing CD28– T cells, were substantially reduced. These results are in line with previous studies suggesting that certain T cell subsets are sequestered away from the peripheral blood during P. falciparum malaria.

Acute P. falciparum malaria is associated with activation of peripheral T cells (1), spontaneous proliferation (2) and increased cytokine production (3,4). However, malaria is also associated with general lymphopenia (5–7). This phenomenon has been explained by apoptosis of peripheral lymphocytes (8,9), reallocation of lymphocytes away from the periphery (10) or both (11). At birth essentially all CD8+ T cells express the co-stimulatory molecule CD28 at their surface, but with increasing age CD8+CD28– T cells accumulate. This increase seems to be the result of viral infection or other immune activation (12). The role of CD28– T cells is not fully understood; however, these are cells that have reached a state of replication senescence (13,14), are potent producers of a number of cytokines (15) and exert potent cytotoxic activity (12). Activated T cells also up-regulate CD95, which predisposes them to apoptosis or activationinduced cell death (AICD) (16,17). The increase in frequencies of CD28– T cells during immune activation and their great potential for cytokine production calls for analysis of this subset in malaria infections, since many cytokines are involved in the pathogenesis of malaria (18–21). In the present study, we investigated CD95 and Bcl-2 as markers of susceptibility to apoptosis and the expression of IFN-γ and IL-4 by CD28– and CD28+ T cells in West African children with acute P. falciparum malaria. Fourteen children were admitted as in-patients to the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana with a diagnosis of P. falciparum malaria. Only patients that could be categorized as having cerebral malaria or uncomplicated malaria according to a set of strict inclusion and exclusion criteria (described elsewhere) were admitted (22). All patients were treated with standard

Keywords apoptosis, CD28, cytokines, malaria, T-cells

Correspondence: Kåre Kemp, PhD, Department of Infectious Diseases M7641, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark (e-mail: [email protected]). Received: 31 January 2002 Accepted for publication: 12 December 2002 © 2002 Blackwell Science Ltd

545

K. Kemp et al.

Parasite Immunology

Table 1 Median and range of percentage of cells expressing CD95, CD28, IFN-γ, IL-4, Bcl-2 or a combination before treatment or 30 days after treatment (n = 14)

CD95a CD28a IFN-γa IL-4a Bcl-2a CD28–IFN-γ+b CD28+IFN-γ+c CD28–IL-4+b CD28+IL-4+c aPercentage

Combination of antibodies used for identification

Acute malaria

Day 30

T-value

P-value

CD3, CD3, CD3, CD3, CD3, CD3, CD3, CD3, CD3,

58·5 (44–72) 78·4 (56–89) 9·4 (5–17) 9·8 (3–19) 75·7 (56–91) 17 (13–31) 9·7 (4–19) 17 (4–29) 7 (2–17)

47·4 (36–69) 78 (57–91) 8·7 (4–16) 4·7 (1–11) 77·8 (66–93) 24 (15–36) 6·2 (3–13) 11·1 (6–26) 3·9 (1–10)

2·5 0·2 0·6 2·6 1 1·7 1·8 2·4 2·3

P = 0·03 P = 0·9 P = 0·6 P = 0·03 P = 0·3 P = 0·1 P = 0·09 P = 0·03 P = 0·03

CD95 CD28 IFN-γ IL-4 Bcl-2 CD28, CD28, CD28, CD28,

IFN-γ IFN-γ IL-4 IL-4

of all CD3+ cells, bpercentage of all CD3+CD28–, cpercentage of all CD3+CD28+.

regimens of chloroquine or artesunate and all recovered fully from their infection. Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep (Nyegaard, Oslo, Norway) density centrifugation. The cells were frozen, stored and transported in liquid nitrogen (23). Before use, the cells were rapidly thawed and washed. The viability of the cells was ascertained by trypan blue exclusion. Previous studies have shown that freezing does not change the number or composition of lymphocyte subsets (24). PBMC were resuspended in RPMI-1640, supplemented with 15% heat-inactivated pooled human serum, 20 IU/mL of penicillin and 20 µg/mL of streptomycin (Gibco, Paisley, UK) and seeded into 24-well multi-dish plates (Nunc, Roskilde, Denmark). Each well contained 1 × 106 PBMC in 1 mL of medium. The cells were cultured at 37°C in a humidified atmosphere with 5% CO2. For intracellular cytokine detection, the cells were incubated with 1·5 µ monensin (Sigma, St. Louis, MO, USA), 1 µ ionomycin and 50 ng /mL PMA for 4 h. For surface staining three-colour flow cytometry was applied using mAb to the determinants listed below as either FITC, phycoerythrin or Cy5 conjugates; CD3 (UCHT1; DAKO, Glostrup, Denmark), CD4 (MT310; DAKO), CD8 (DK25; DAKO), CD28 (CD28; DAKO), CD95 (M019751, Pharmingen, CA, USA). Intracellular staining methods for IFN-γ or IL-4 (Pharmingen, CA, USA), anti-Bcl-2 (Pharmingen) and/ or anti-CD4 (MT310; DAKO) were as described previously (25). All samples were analysed on a FACScan flow cytometer (Becton Dickinson). Analysis of data was carried out using Winlist software (Verity, Topsham, ME). The absolute number of lymphocytes in each subset was calculated by multiplying the total lymphocyte count by the proportion of lymphocytes in that subset. Groups were compared by Student’s t-test for paired data. Values of P < 0·05 were considered significant. Preliminary

546

data analysis revealed that there were no significant differences in the parameters investigated between children with cerebral malaria and children with uncomplicated malaria, and data from both groups are consequently presented together. Thirty days following treatment there was no significant difference in the parameters investigated between patients and a group of healthy children from a nearby community (data not shown). To investigate the activation status of T cells in the periphery, the frequency of CD95+ (Fas) T cells was measured. Malaria patients showed increased percentages of CD95+CD3+ T cells on admission when compared to 30 days following treatment (P = 0·03) (Table 1). The proportion of cytokine-producing cells was also higher before treatment; however, this difference was only significant with respect to IL-4 producing cells (P = 0·03); no significant differences were found in respect to Bcl-2 expression (Table 1). Intracellular staining for IL-4 and IFN-γ was performed to investigate the Th1/ Th2 dichotomy within the CD28+ and CD28– T cell populations during acute malaria. Children had significantly higher frequencies of both IL-4 producing CD28– T cells and IL-4 producing CD28+ T cells on admission when compared to 30 days after treatment (P = 0·03) (Table 1). The frequency of IFN-γ producing CD28+ T cells was higher during acute disease, but this was not statistically significant. In contrast, the proportion of IFN-γ producing CD28– T cells was lower during acute disease, but again this difference was not statistically significant. Since acute malaria is associated with lymphopenia (Table 2) we hypothesized that the absolute numbers of IFN-γ producing CD28– T cells would be significantly lower during acute disease. This was confirmed by calculations of the absolute numbers of T cells and CD28–IFN-γ+ T cells (Table 2). The absolute number of IL-4 producing CD28– T cells was slightly higher during acute disease, although this difference was not statistically significant. © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 545–548

Volume 24, Number 11/12, November/December 2002

Table 2 Median and range of absolute counts of lymphocyte subsets (counts /µL) (n = 14)

CD3+ CD3+CD28+IFN-γ+ CD3+CD28–IFN-γ+ CD3+CD28+IL-4+ CD3+CD28–IL-4+

The low number of IFN-γ producing CD28– T cells in acutely ill malaria patients may be a consequence of apoptosis among T lymphocytes, especially since CD28– T cells are at a state of replication senescence and prone to apoptosis (13,14). Indeed this study shows an increase in the number of pre-apoptotic (CD95+) T cells in acute malaria, and a majority of the CD28– T cells expressed CD95 (not shown). However, we and others have suggested that some T cells are sequestered during acute malaria (7), leading to loss of certain T cell subsets from the periphery (10,11). High levels of circulating IFN-γ in the plasma of acute malaria patients (18) suggest that IFN-γ producing cells may indeed be sequestered away from the peripheral blood. Thus, although the present study shows an increased percentage of activated T lymphocytes in the circulation of malaria patients, absolute T cell numbers are substantially reduced. Cell subsets that are important for outcome of the disease in terms of production of pro-inflammatory cytokines may be sequestered elsewhere in the body.

ACKNOWLEDGEMENTS This work was supported by grants from the Danish International Development Agency (RUF and the ENRECA programmes), the Danish Biotechnology Programme and the Novo Nordisk Foundation. G. Grauert is thanked for excellent technical support.

REFERENCES 1 Elhassan IM, Hviid L, Satti G et al. Evidence of endothelial inflammation, T cell activation, and T cell reallocation in uncomplicated Plasmodium falciparum malaria. Am J Trop Med 1994; 51: 372–379. 3 Worku S, Troye-Blomberg M, Christensson B, Bjorkman A & Fehniger T. Activation of T cells in the blood of patients with acute malaria: proliferative activity as indicated by Ki-67 expression. Scand J Immunol 2001; 53: 296–301. 4 Winkler S, Willheim M, Baier K et al. Frequency of cytokineproducing T cells in patients of different age groups with Plasmodium falciparum malaria. J Infect Dis 1999; 179: 209– 216. 5 Winkler S, Willheim M, Baier K et al. Reciprocal regulation of Th1- and Th2-cytokine-producing T cells during clearance of parasitemia in Plasmodium falciparum malaria. Infect Immun 1998; 66: 6040–6044. © 2002 Blackwell Science Ltd, Parasite Immunology, 24, 545 –548

Loss of CD28 – T cells in acute P. falciparum malaria

Acute malaria

Day 30

T-value

P-value

660 (372–929) 49 (13–97) 19 (2–23) 36 (6–44) 17 (1–56)

981 (451–1487) 48 (9–101) 47 (1–80) 29 (7–32) 9 (0–23)

5·7 0·2 2·3 0·2 2

P < 0·001 P = 0·9 P = 0·04 P = 0·8 P = 0·05

6 Wells RA, Pavanand K, Zolyomi S, Permpanich B & MacDermott RP. Loss of circulating T lymphocytes with normal levels of B and ‘null’ lymphocytes in Thai adults with malaria. Clin Exp Immunol 1979; 35: 202–209. 7 Wyler DJ. Peripheral lymphocyte subpopulations in human Plasmodium falciparum malaria. Clin Exp Immunol 1976; 23: 471–476. 8 Hviid L, Kurtzhals JAL, Goka BQ, Oliver-Commey JO, Nkrumah FK & Theander TG. Rapid reemergence of T cells into peripheral circulation following treatment of severe and uncomplicated Plasmodium falciparum malaria. Infect Immun 1997; 65: 1090–1093. 9 Baldé AT, Sarthou J-L & Roussilhon C. Acute Plasmodium falciparum infection is associated with increased percentages of apoptotic cells. Immunol Lett 1995; 46: 59–62. 10 Toure-Balde A, Sarthou JL, Aribot G et al. Plasmodium falciparum induces apoptosis in human mononuclear cells. Infect Immun 1996; 64: 744–750. 11 Hviid L, Theander TG, Abdulhadi NH, Abu-Zeid YA, Bayoumi RA & Jensen JB. Transient depletion of T cells with high LFA-1 expression from peripheral circulation during acute Plasmodium falciparum malaria. Eur J Immunol 1991; 21: 1249–1253. 12 Kemp K, Akanmori BD, Adabayeri V, et al. Cytokine production and apoptosis among T cells from patients under treatment for Plasmodium falciparum malaria. Clin Exp Immunol 2002; 127: 151–157. 13 Fagnoni FF, Vescovini R, Mazzola M et al. Expansion of cytotoxic CD8+. Immunology 1996; 88: 501–507. 14 Effros RB, Allsopp R, Chiu CP et al. Shortened telomeres in the expanded CD28–CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis. AIDS 1996; 10: F17–F22. 15 Monteiro J, Batliwalla F, Ostrer H & Gregersen PK. Shortened telomeres in clonally expanded CD28 –CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts. J Immunol 1996; 156: 3587–3590. 16 Labalette M, Leteurtre E, Thumerelle C, Grutzmacher C, Tourvieille B & Dessaint JP. Peripheral human CD8(+)CD28(+) T lymphocytes give rise to CD28(–) progeny, but IL- 4 prevents loss of CD28 expression. Int Immunol 1999; 11: 1327–1336. 17 Alderson MR, Armitage RJ, Maraskovsky E et al. Fas transduces activation signals in normal human T lymphocytes. J Exp Med 1993; 178: 2231–2235. 18 Ju S-T, Panka DJ, Cui H et al. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 1995; 373: 444 – 448. 19 Rhodes-Feuillette A, Bellosguardo M, Druilhe P et al. The interferon compartment of the immune response in human malaria: II. Presence of serum-interferon gamma following the acute attack. J Interferon Res 1985; 5: 169–178. 20 Grau GE, Taylor TE, Molyneux ME et al. Tumor necrosis factor and disease severity in children with falciparum malaria. New Engl J Med 1989; 320: 1586–1591.

547

K. Kemp et al.

21 Kwiatkowski D, Hill AV, Sambou I et al. TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 1990; 336: 1201–1204. 22 Wenisch C, Parschalk B, Narzt E, Looareesuwan S & Graninger W. Elevated serum levels of IL-10 and IFN-γ in patients with acute Plasmodium falciparum malaria. Clin Immunol Immunopathol 1995; 74: 115–117. 23 Kurtzhals JA, Adabayeri V, Goka BQ et al. Low plasma concentrations of interleukin 10 in severe malarial anaemia compared with cerebral and uncomplicated malaria. Lancet 1998; 351: 1768–1772.

548

Parasite Immunology

24 Hviid L, Albeck G, Hansen B, Theander TG & Talbot A. A new portable device for automatic controlled-gradient cryopreservation of blood mononuclear cells. J Immunol Meth 1993; 157: 135–142. 25 Kemp K, Akanmori BD & Hviid L. West African donors have high percentages of activated cytokine producing T cells that are prone to apoptosis. Clin Exp Immunol 2001; 126: 69. 26 Kemp K & Bruunsgaard H. Identification of IFN-gammaproducing CD4+ T cells following PMA stimulation. J Interferon Cytokine Res 2001; 21: 503–506.

© 2002 Blackwell Science Ltd, Parasite Immunology, 24, 545–548