The importance of dendritic cells (DCs) for the initiation and regulation of immune responses not only to foreign organisms but also to the self has raised considerable interest in the qualitative and quantitative analysis of these cells in various human diseases.Plasmodium falciparum malaria is characterized by the poor induction of long-lasting protective immune responses. This study, therefore, investigated the percentage of peripheral blood DCs as lineage marker–negative and HLA-DR+ or CD83+cells in healthy children and in children suffering from acute malaria in Kilifi, Kenya. Comparable percentages of CD83+ DCs were found in peripheral blood of healthy children and children with malaria. However, the percentage of HLA-DR+ peripheral blood DCs was significantly reduced in children with malaria. The results suggest that a proportion of peripheral blood DCs may be functionally impaired due to the low expression of HLA-DR on their surface.

Dendritic cells (DCs) are central for the induction of immune responses to pathogens infecting the human host because they transport antigen from the periphery to lymphoid tissue in response to inflammatory signals. Here, they activate naive T cells and boost memory responses.1 It is therefore not surprising that many pathogens have evolved mechanisms to subvert the function of this important cell. In endemic areas, infection with Plasmodium falciparum results in a wide range of outcomes from asymptomatic infection to severe disease and death. Acquisition of immunity is through exposure and therefore age-related and occurs only after several years of constant exposure to mild disease and probably never to asymptomatic infection.2,3 Among other mechanisms of immune evasion, P falciparum–infected erythrocytes (iRBCs) modulate the maturation and function of monocyte-derived DCs in vitro.4 Given the central role of DCs in the induction of immune responses, we analyzed the number of circulating peripheral blood DCs ex vivo in children with malaria and in healthy children in Kilifi, Kenya.5 

Study population

The study was performed at the district hospital in Kilifi, Kenya, between July and September 1999. Children suffering from mild malaria were treated in the outpatient department, whereas children suffering from severe malaria who were prostrated, hypoglycemic, in respiratory distress, or severely anemic were admitted to the high-dependency ward.6 Children who also suffered from diseases other than malaria were excluded from analysis. A control group of afebrile children with negative blood films were recruited into the study during a cross-sectional survey. The study was approved by the National Ethical Committee, Kenya.

Analysis of blood samples

Blood (2 mL) was obtained from each child and a differential blood count performed. Interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in plasma were measured in triplicate by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's recommendations (R & D Systems, Abingdon, United Kingdom). The sensitivity of the assay was 15 pg/mL for TNF-α and for IL-10. Peripheral blood mononuclear cells (PBMCs) were separated and diluted to 1 × 107/mL. A proportion of cells was incubated for 24 hours in RPMI supplemented with 10% human AB serum, 2 mM glutamine, 50 μg/mL kanamycin, and 10 mM HEPES to induce surface expression of CD83.5,7 For the detection HLA-DR+ or CD83+ DCs, cells were incubated in duplicate with a cocktail of lineage-specific monoclonal antibodies against CD3 (UCHT1), CD19 (TÜK4), CD14 (HD37), CD16 (DJ130c), and CD56 (MOC1) for 30 minutes at 4°C, and bound antibodies were detected using R-phycoerythrin (RPE)–conjugated goat anti–mouse immunoglobulin antibody. Cells were incubated for 10 minutes in 10% mouse serum before addition of the fluorescein isothiocyanate (FITC)–conjugated isotype control, anti–HLA-DR (CR3/43), or anti-CD83 antibody (HB-15e, Pharmingen, Oxford, United Kingdom).5 For detection of CD34+progenitor cells, cells were stained with anti-CD45 (T29/33, 2B11) and FITC-conjugated anti-CD34 antibodies (BIRMA-K3).8 If not stated otherwise, antibodies were purchased from Dako (Ely, United Kingdom). At least 50 000 events were analyzed by flow cytometry (Epics XL, Beckmann-Coulter, High Wycombe, United Kingdom). Samples that had fewer than 0.02% positive events were regarded as being below the detection limit.

Statistical analysis

Parameters were compared between groups of children using a Mann-Whitney U test and correlated within groups of children using the Spearman rank correlation test. The agreement of the 2 detection methods for peripheral blood DCs was examined according to a method by Altman.9 

Circulating peripheral blood DCs were readily detected in the blood of healthy control children both as a population of HLA-DR+ DCs immediately after purification of PBMCs and as CD83+ DCs after a period of culture (Figure1). Both detection methods gave a similar overall distribution with a median percentage of 1.15% (interquartile range [IQR] 0.53-1.8) for HLA-DR+ DCs and a median percentage of 1.1% (IQR 0.48-1.8) for CD83+ DCs. Within the group of healthy children, we found good agreement between the 2 methods with a mean individual difference of 0.1% (SD 1%).9 The percentage of HLA-DR+ and CD83+ peripheral blood DCs correlated with each other (Spearman rho = 0.557, P < .01) but not with the age of the child (CD83+, rho = 0.085; HLA-DR+, rho = −0.015) or with white blood cells (CD83+, rho = 0.0.07; HLA-DR+, rho = 0.215; Table1). These results imply that both methods detected an overlapping population of DCs rather than distinct subsets. The methods did not distinguish between myeloid and plasmacytoid DCs because both populations constitutively express HLA-DR and, after a period of in vitro culture, CD83.10 11 

Fig. 1.

Identification and distribution of peripheral blood DCs.

(A) PBMCs were gated and at least 50 000 events were acquired per sample. B cells, T cells, monocytes, and natural killer cells were detected with a cocktail of lineage-specific antibodies (lineage-marker) in FL-2. DCs were detected as lineage marker–negative cells with an isotype control antibody or anti–HLA-DR antibody in FL-1. Shown are examples of HLA-DR+ DCs in one healthy child (upper panel) and in 2 children suffering from severe malaria (middle and lower panels). (B) The boxplots indicate median and 25% and 75% percentiles for the percentage of CD83+ cells or HLA-DR+ DCs in healthy children and in children with severe or mild malaria. Outliers are indicated by open circles and extreme values are indicated by stars.

Fig. 1.

Identification and distribution of peripheral blood DCs.

(A) PBMCs were gated and at least 50 000 events were acquired per sample. B cells, T cells, monocytes, and natural killer cells were detected with a cocktail of lineage-specific antibodies (lineage-marker) in FL-2. DCs were detected as lineage marker–negative cells with an isotype control antibody or anti–HLA-DR antibody in FL-1. Shown are examples of HLA-DR+ DCs in one healthy child (upper panel) and in 2 children suffering from severe malaria (middle and lower panels). (B) The boxplots indicate median and 25% and 75% percentiles for the percentage of CD83+ cells or HLA-DR+ DCs in healthy children and in children with severe or mild malaria. Outliers are indicated by open circles and extreme values are indicated by stars.

Close modal
Table 1.

General description of study population

Severe malaria
n = 33
Mild malaria
n = 34
Community
controls
n = 34
Age (mo) 33.9 40.2 66.9 
 (13.4-57) (27.9-73.1) (30-95)  
Axillary temperature (°C) 38* 36.5 36.5 
 (37.1-38.7) (36.3-38.9) (36.3-36.7)  
WBCs (× 106/mL) 12 6.9  9.9 
 (9.4-17.65) (5.9-11.5) (6.7-11.65)  
PBMCs (× 106/mL) 5.3 3.7  6.3 
 (4.1-8.8) (3.0-5.2) (3.8-8.1)  
Granulocytes (× 106/mL) 6.0 3.3  3  
 (3.7-9.4) (2.1-6.2) (2.0-4.1)  
RBCs (× 109/mL) 2.7 3.7  4.4 
 (2.1-3.7) (2.9-4.3) (4-4.7)  
Hemoglobin (mg/dL) 7.8 9* 10.1 
 (4.9-9.6) (7.8-10.1) (8.9-11)  
Parasitemia (no./μL) 140 620 22 080  0  
 (5 964-411 875) (11 847-46 954)  
TNF-α (pg/mL) 130 98 52  
 (85-180) (83-152) (46-84)  
IL-10 (pg/mL) 225 157  0  
 (87-2 283) (60-459)  
Severe malaria
n = 33
Mild malaria
n = 34
Community
controls
n = 34
Age (mo) 33.9 40.2 66.9 
 (13.4-57) (27.9-73.1) (30-95)  
Axillary temperature (°C) 38* 36.5 36.5 
 (37.1-38.7) (36.3-38.9) (36.3-36.7)  
WBCs (× 106/mL) 12 6.9  9.9 
 (9.4-17.65) (5.9-11.5) (6.7-11.65)  
PBMCs (× 106/mL) 5.3 3.7  6.3 
 (4.1-8.8) (3.0-5.2) (3.8-8.1)  
Granulocytes (× 106/mL) 6.0 3.3  3  
 (3.7-9.4) (2.1-6.2) (2.0-4.1)  
RBCs (× 109/mL) 2.7 3.7  4.4 
 (2.1-3.7) (2.9-4.3) (4-4.7)  
Hemoglobin (mg/dL) 7.8 9* 10.1 
 (4.9-9.6) (7.8-10.1) (8.9-11)  
Parasitemia (no./μL) 140 620 22 080  0  
 (5 964-411 875) (11 847-46 954)  
TNF-α (pg/mL) 130 98 52  
 (85-180) (83-152) (46-84)  
IL-10 (pg/mL) 225 157  0  
 (87-2 283) (60-459)  

Shown are the median and interquartile range. Parameters for severe and mild disease were compared to healthy controls using the Mann-Whitney U test.

WBCs indicates white blood cells.

*

P < .001.

P < .01.

P < .0001.

We found no evidence for a difference between the percentages of CD83+ DCs in children with mild or severe malaria and healthy children (median and IQR: severe malaria 0.7%, 0.15-1.8,P = .153; mild malaria 0.7, 0.26-1.8,P = .276; Figure 1B). This observation is surprising because it has been reported that infectious diseases in both humans and mice induce the rapid migration of DCs into lymphoid tissue.5 12-15 During acute malaria, DCs may either not migrate into the spleen or they may be subjected to a higher turnover, which, in its steady state, would indicate normal percentages of circulating blood DCs. The latter hypothesis would be compatible with the observation that children with malaria were more likely to have detectable levels of circulating CD34+ progenitor cells (severe malaria, 77%; mild malaria, 82%; healthy children, 53%; Pearson χ2 test on 2 df = 11.93,P = .002).

However, the percentages of HLA-DR+ DCs were significantly lower in children with severe and mild malaria compared to healthy children (median and IQR: severe malaria 0.19%, 0.08-0.68,P < .0001; mild malaria 0.36, 0.07-1,P < .002; Figure 1B). This difference was independent of age (rho = 0.004) and parameters of malarial disease (Table 1) such as granulocyte counts (rho = −0.24), parasitemia (rho = 0.007), or the plasma concentration of IL-10 (rho = 0.044) and TNF-α (rho = −0.075). Furthermore, the mean fluorescence intensity of HLA-DR+ DC was reduced in children with malaria (median and IQR: malaria 89, 69-107; controls 249, 223-392;P < .0001). Given that CD83+ and HLA-DR+ DCs are an overlapping population, we suggest that a proportion of peripheral blood DCs in children with malaria is functionally impaired. This observation is consistent with our in vitro studies demonstrating that adhesion of infected erythrocytes to DCs modulates their maturation and function that may have consequences for the initiation and maintenance of immune responses.4Studies both in human falciparum malaria and in mouse malaria have shown that the induction of primary immune responses during acute malarial disease can be impaired.16-19 By contrast, antibody responses to a diverse number of parasite antigens are readily induced although only a subset of these seem to be associated with protection from disease.20-22 However, antibody responses to both nonvariant and variant-specific targets of the infecting parasite can be short-lived, indicating that here maintenance of humoral immune response may be disturbed.23 24 

Clearly, our observation of altered DC phenotype in the peripheral blood of children with acute malaria warrants further investigation. Considering the high surface expression of CD36 on plasmacytoid DCs10 and the ability of iRBCs to bind to CD36, it will be of particular interest to differentiate quantitatively and qualitatively whether all DC subsets are affected similarly during acute malaria and after the onset of treatment. Such studies may contribute to the understanding of mechanisms of immune evasion by iRBCs.

This study is published with permission of the director of the Kenyan Medical Research Institute.

Supported by the Sir E. P. Abraham Trust, University of Oxford (B.C.U.) and the Kenyan Medical Research Institute. K.M. is a Wellcome Trust Senior Clinical Research Fellow.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Banchereau
J
Briere
F
Caux
C
et al
Immunobiology of dendritic cells.
Annu Rev Immunol.
18
2000
767
811
2
McGregor
IA
Malarial immunity: current trends and prospects.
Ann Trop Med Parasitol.
81
1987
647
656
3
Gupta
S
Snow
RW
Donnelly
CA
Marsh
K
Newbold
CI
Immunity to non-cerebral severe malaria is acquired after one or two infections.
Nat Med.
5
1999
340
343
4
Urban
BC
Ferguson
DJP
Pain
A
et al
Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells.
Nature.
400
1999
73
77
5
Fearnley
DB
Whyte
LF
Carnoutsos
SA
Cook
AH
Hart
DN
Monitoring human blood dendritic cell numbers in normal individuals and in stem cell transplantation.
Blood.
93
1999
728
736
6
Marsh
K
Forster
D
Waruiru
C
et al
Indicators of life-threatening malaria in African children.
N Engl J Med.
332
1995
1399
1404
7
Zhou
LJ
Tedder
TF
Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily.
J Immunol.
154
1995
3821
3835
8
Gratama
JW
Orfao
A
Barnett
D
et al
Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells. European Working Group on Clinical Cell Analysis.
Cytometry.
34
1998
128
142
9
Altman
DG
Some common problems in medical research: method comparison studies.
Practical Statistics for Medical Research. London
Altman
DG
1991
396
403
Chapman & Hall
United Kingdom
10
Cella
M
Jarrossay
D
Facchetti
F
et al
Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon.
Nat Med.
5
2000
919
923
11
Cella
M
Facchetti
F
Lanzavecchia
A
Colonna
M
Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization.
Nat Immunol.
1
2000
305
310
12
Patterson
S
Robinson
SP
English
NR
Knight
SC
Subpopulations of peripheral blood dendritic cells show differential susceptibility to infection with a lymphotropic strain of HIV-1.
Immunol Lett.
66
1999
111
116
13
Austyn
JM
Kupiec-Weglinski
JW
Hankins
DF
Morris
PJ
Migration patterns of dendritic cells in the mouse: homing to T-cell dependent areas of spleen, and binding within marginal zone.
J Exp Med.
167
1988
646
651
14
De Smedt
T
Pajak
B
Muraille
E
et al
Positive and negative regulation of dendritic cell function by lipopolysaccharide in vivo.
Adv Exp Med Biol.
417
1997
535
540
15
Kanath
AT
Pooley
J
O'Keeffe
MA
et al
The development, maturation, and turnover rate of mouse spleen dendritic cell population.
J Immunol.
165
2000
6762
6770
16
McGregor
IA
Barr
M
Antibody responses to tetanus toxoid inoculation in malarious and non-malarious Gambian Children.
Trans R Soc Trop Med Hyg.
56
1962
364
367
17
Williamson
WA
Greenwood
BM
Impairment of the immune response to vaccination after acute malaria.
Lancet.
7
1978
1328
1329
18
Greenwood
BM
Bradley
AK
Blakebrough
IS
Whittle
HC
Marshall
TF
Gilles
HM
The immune response to a meningococcal polysaccharide vaccine in an African village.
Trans R Soc Trop Med Hyg.
74
1980
340
346
19
Whittle
HC
Brown
J
Marsh
K
et al
T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria.
Nature.
312
1984
449
450
20
Marsh
K
Howard
RJ
Antigens induced on erythrocytes by P. falciparum: expression of diverse and conserved determinants.
Science.
231
1986
150
153
21
Bull
PC
Lowe
BS
Kortok
M
Molyneux
CS
Newbold
CI
Marsh
K
Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria.
Nat Med.
4
1998
358
360
22
Guevara Patino
JA
Holder
AA
McBride
JS
Blackman
MJ
Antibodies that inhibit malaria merozoite surface protein-1 processing and erythrocyte invasion are blocked by naturally acquired human antibodies.
J Exp Med.
186
1997
1689
1699
23
Giha
HA
Staalsoe
T
Dodoo
D
et al
Nine-year longitudinal study of antibodies to variant antigens on the surface of Plasmodium falciparum-infected erythrocytes.
Infect Immun.
67
1999
4092
4098
24
Cavanagh
DR
Elhassan
IM
Roper
C
et al
A longitudinal study of type-specific antibody responses to Plasmodium falciparum merozoite surface protein-1 in an area of unstable malaria in Sudan.
J Immunol.
161
1998
347
359

Author notes

Britta C. Urban, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom; e-mail: burban@hammer.imm.ox.ac.uk.

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