ArticlePDF AvailableLiterature Review

Immunological processes in malaria pathogenesis

October 2005
Nature Reviews Immunology 5(9):722-35

October 2005
5(9):722-35

DOI:10.1038/nri1686

Source
PubMed

Authors:

Louis Schofield

James Cook University

Georges E Grau

The University of Sydney

Malaria is possibly the most serious infectious disease of humans, infecting 5-10% of the world's population, with 300-600 million clinical cases and more than 2 million deaths annually. Adaptive immune responses in the host limit the clinical impact of infection and provide partial, but incomplete, protection against pathogen replication; however, these complex immunological reactions can contribute to disease and fatalities. So, appropriate regulation of immune responses to malaria lies at the heart of the host-parasite balance and has consequences for global public health. This Review article addresses the innate and adaptive immune mechanisms elicited during malaria that either cause or prevent disease and fatalities, and it considers the implications for vaccine design.

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Content may be subject to copyright.

*The Walter and Eliza Hall

Institute of Medical

Research, 1G Royal Parade,

Parkville, Victoria 3050,

Australia.

‡

Université de la

Méditerranée, Centre

National de la Recherche

Scientifique, Unité Mixte

de Recherche 6020,

Immunopathology Group,

Faculty of Medicine,

Institut Fédératif de

Recherche 48, 27 boulevard

Jean Moulin, F-13385

Marseille, France.

Correspondence to L.S.

e-mail:

schofield@wehi.edu.au

doi:10.1038/nri1686

Malaria is transmitted to vertebrate hosts, such as mice,

monkeys and humans, by the bite of female Anopheles

mosquitoes that are infected with protozoan parasites

of the genus Plasmodium. The inoculated sporozoite

stage is transient and causes no pathology. Within

a few minutes, it infects liver cells and undergoes a

period of intracellular replication, which is also clini-

cally silent. After liver-stage replication is complete,

the parasite initiates blood-stage infection, which

is the main cause of disease

(FIG. 1). There are four

Plasmodium species that infect humans. Plasmodium

ovale typically causes a relatively benign infection.

Plasmodium malariae is also frequently clinically

silent, although an immune-complex-associated

glomerulonephropathy can develop following chronic

infection. Although it is rarely fatal, Plasmodium vivax

is a common cause of acute febrile illness, especially

in Asia, South America and Oceania, and it might

contribute to anaemia. However, most cases of severe

disease and most deaths are caused by the blood-stage

cycle of Plasmodium falciparum, which is endemic in

most of sub-Saharan Africa and throughout most of

the tropics.

Worldwide, most infections with malaria-causing

agents are clinically silent, reflecting the ability of

adaptive immune mechanisms to prevent disease.

In non-immune individuals, however, infections are

more clinically overt, and a minority of these can

become severe or life threatening, manifesting a range

of discrete and overlapping disease syndromes of

complex aetiologies. Those dying of malaria can have

single-organ, multiple-organ or systemic involvement

TABLE 1. Overall patterns of disease depend markedly

on the age and the previous immunological experi-

ence of the host

. In areas of high malaria transmis-

sion, the burden of disease is borne by infants and

young children; life-threatening disease in this setting

typically consists of metabolic acidosis (which leads to

respiratory distress), cerebral malaria (CM) and severe

malarial anaemia (SMA). However, in areas of lower

transmission, primary infections might occur in adult-

hood, in which severe disease more frequently involves

additional disturbances, such as renal failure, pulmo-

nary oedema, shock and jaundice. So, transmission

dynamics and host age are important determinants of

disease, together with host genetics and immunological

responses (discussed later).

The diversity of syndromes

TABLE 1 seems to con-

found the identification of unifying mechanisms of

disease. However, the studies reviewed here generally

support a scheme in which several important malaria

syndromes might arise from the intersection of a few

basic processes: the site-specific localization of para-

sitized red blood cells (PRBCs) among target organs;

IMMUNOLOGICAL PROCESSES

IN MALARIA PATHOGENESIS

Louis Schofield* and Georges E. Grau

‡

Abstract | Malaria is possibly the most serious infectious disease of humans, infecting 5–10%

of the world’s population, with 300–600 million clinical cases and more than 2 million deaths

annually. Adaptive immune responses in the host limit the clinical impact of infection and

provide partial, but incomplete, protection against pathogen replication; however, these

complex immunological reactions can contribute to disease and fatalities. So, appropriate

regulation of immune responses to malaria lies at the heart of the host–parasite balance and

has consequences for global public health. This Review article addresses the innate and

adaptive immune mechanisms elicited during malaria that either cause or prevent disease

and fatalities, and it considers the implications for vaccine design.

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the local and systemic action of bioactive parasite

products, such as toxins, on host tissues; the local

and systemic production of pro-inflammatory and

counter-regulatory cytokines and chemokines by the

innate and adaptive immune systems in response to

parasite products; and the activation, recruitment

and infiltration of inflammatory cells. According

to this view, diverse organ-specific or systemic dis-

ease syndromes are end-stage processes of atypical

inflammatory cascades that are initiated in target

organs by pathogen products and are maintained by

infiltrating cells through positive-feedback cycles. In

most cases, homeostasis corrects the cascade effect,

and responses are adequately downregulated. In severe

disease, however, a ‘run-away’ effect can ensue, with

fatal consequences. Appropriate regulation of immune

responses might therefore be a key to healthy out-

comes, and understanding these processes might aid

in the development of vaccine-based interventions.

Initiation of malaria-associated syndromes

Site-specific localization of PRBCs. As blood-stage

parasites mature through the 48-hour replicative cycle,

avoiding passage through the spleen is an essential

survival strategy, because this immunological effector

organ

efficiently filters PRBCs from the bloodstream.

Erythrocyte membrane protein 1 (

EMP1) is the name

given collectively to members of a family of variant

cell-surface proteins that are encoded by P. f a l c i p a r u m

and enable PRBCs to engage multiple receptors — such

as intercellular adhesion molecule 1 (

ICAM1), vascu-

lar cell-adhesion molecule 1 (VCAM1), CD31,

CD36,

Figure 1 | The life cycle of Plasmodium falciparum. Mosquitoes that carry the malaria-causing parasite Plasmodium falciparum

inject a small number of infectious sporozoites into the bloodstream while feeding. Within a few minutes, they are carried to the liver,

where they invade and replicate in liver cells. Then, 10–12 days later, thousands of daughter merozoites are released back into the

bloodstream and enter red blood cells (RBCs). The parasites are carried around the circulation within RBCs, but as they grow, they

express adherent ligands — such as P. falciparum erythrocyte membrane protein 1 — that enable the maturing parasite to bind

receptors expressed by endothelial cells that line the blood vessels in the deep vascular beds of organs such as the brain, lungs and

placenta. After 48 hours, the parasitized RBCs (PRBCs) rupture and release more daughter merozoites, thereby perpetuating

and promoting the blood-stage cycle. The presence of the parasite and the invasion of RBCs might not be sufficient to account

for disease; instead, the release of bioactive parasite molecules and an inappropriately regulated host immune response could be

the main causes of fatal pathogenesis, which occurs in only a minority of patients. Some merozoites differentiate into gametocytes,

which, when taken up by another feeding mosquito, perpetuate the sexual cycle in the insect.

Blood vessel

Umbilical

cord

PRBC

Mosquito

Brain

Placenta

Merozoites

enter RBC

RBC

schizont

Some merozoites

develop into

gametocytes

Asexual reproductive

stages in RBC

Sporozoites

released into

bloodstream

Liver cells

release

merozoites

Placental malaria

Cerebral malaria

Mosquito injects

sporozoites

Sequestration of

PRBCs on placental

endothelium

Inflammatory response to

PRBCs and parasite toxins

PRBC Platelet Leukocyte

Endothelial

cell

Vascular

lumen

Sporozoites rapidly

enter liver cells

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CHAGAS’ DISEASE

A disease that is caused by

Trypanosoma cruzi. In chronic

cases, it is associated with

autoimmune damage to

various organs.

thrombospondin, endothelial-cell selectin (E-selectin),

chondroitin sulphate A (CSA) and hyaluronic acid

— that are expressed by vascular endothelial cells

in deep-organ microvascular beds. Binding to these

receptors by cell-surface P. falciparum EMP1 seques-

ters parasites so that they are removed from the circu-

lation and, consequently, do not travel to the spleen.

Although this is advantageous for the survival of the

parasite, this strategy has the pathological consequence

of concentrating parasites in various target organs, and

the precise locations depend on the differential expres-

sion of the various P. falciparum EMP1 members and

their diverse endothelial-cell-expressed receptors.

Production of bioactive parasite products. As

sequestered parasites mature, they produce a vari-

ety of bio active molecules that either upregulate or

downregulate pathogenic processes, largely through

their effects on the innate immune system

TABLE 2.

Immune responses to infectious insults are mainly

initiated by the interaction of pathogen-associated

molecular patterns (PAMPs) with receptors expressed

by host cells. For viruses, bacteria and yeast, PAMPs

include modified lipids (such as bacterial lipopoly-

saccharides), carbohydrates (such as yeast zymosan),

proteins (such as flagellin) and nucleic acids (such

as unmethylated CpG-motif-containing DNA and

double-stranded RNA). Many studies implicate

glycosylphosphatidylinositol (GPI) of P. falciparum

as a malaria PAMP and as a toxin. Purified GPI

induces the expression of many genes that are impli-

cated in malaria pathogenesis: for example, genes

that encode pro-inflammatory cytokines — such

as tumour-necrosis factor (

TNF), interleukin-1

(

IL-1) and IL-12 REFS 36 — inducible nitric-oxide

synthase

, and various adhesion molecules that

are expressed at the surface of the vascular endo-

thelium and are recognized by P. falciparum

EMP1

REF. 8, which increases endothelial-cell binding by

PRBCs

. In a sepsis–shock model, GPI alone is suf-

ficient to cause symptoms that are similar to those

of acute malaria, such as transient pyrexia, hypo-

glycaemia and death of recipients as a consequence

of TNF-mediated coagulopathy, as seen in the

malarial shock-like syndrome

TABLE 1. The GPIs

from Trypanosoma brucei, Trypanosoma cruzi and

Toxoplasma gondii all have similar properties to the

GPI from P. falciparum

9–11

, and this might account

for some pathogenic features of trypanosomiasis,

CHAGAS’ DISEASE and toxoplasmosis.

Other potential P. f a l c i p a r u m PAMPs include phos-

phorylated, non-peptidic antigens, to which γδ T cells

respond with slow kinetics

, and haemozoin, the insol-

uble, crystalline residue of parasite-mediated haemo-

globin digestion, which is long-lived and accumulates in

phagocytes. Haemozoin has interesting, although seem-

ingly contradictory, bioactivities. It has been reported to

induce

or inhibit

dendritic cell (DC) maturation and

to induce either the production of the T helper 1 (T

cytokines TNF

and IL-12 REF. 13 or the T

2 cytokine

IL-10. It also inhibits general proliferative responses by

human leukocytes

. In addition, it has been shown to

promote monocyte and macrophage dysfunction, by

impairing phagocytosis and the expression of MHC

class II molecules, CD11c and ICAM1

REF. 17. Overall,

haemozoin seems to be highly immunosuppressive

18,19

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Table 1 | Severe and fatal disease syndromes in malaria

Syndrome Clinical features Possible sequence or mechanism of disease

Cerebral malaria Sustained impaired consciousness, coma,

long-term neurological sequelae

Cerebral parasite sequestration; bioactive GPI; pro-inflammatory

cytokine cascade; endothelial-cell activation; natural killer T-cell

activation; T

1/T

2-cell balance; chemokine production;

monocyte, macrophage and neutrophil recruitment; platelet

and fibrinogen deposition; CD4

, CD8

and γδ T-cell involvement;

IFN-γ production; neurological metabolic derangements; possibly

hypoxia

Placental malaria Placental insufficiency, low birth weight,

premature delivery, loss of fetus

Plasmodium falciparum EMP1-mediated binding to placental

endothelium and syncytiotrophoblast through chondroitin

sulphate A and hyaluronic acid; cytokine production; chemokine-

mediated recruitment and infiltration of monocytes; intravascular

macrophage differentiation

Severe malarial anaemia Pallor, lethargy, haemoglobin level of 4–6 g

per 10 ml

Erythropoietic suppression by toxins and cytokines; increased

RBC destruction, owing to parasitization, RBC alterations,

complement and immune complex or antigen deposition,

erythrophagocytosis, splenic hyperphagism, CD4

T cells,

1/T

2 cytokine balance (TNF and IFN-γ versus IL-10)

Metabolic acidosis Respiratory distress, deep breathing

(Kussmaul breathing), hypovolaemia

Molecular mechanisms unknown. Possibly widespread parasite

sequestration; bioactive toxins; increased vascular permeability;

reduced tissue perfusion; anaemia; pulmonary airway obstruction;

hypoxia; increased host glycolysis; repressed gluconeogenesis.

Some overlap with shock-like syndrome

Shock-like syndrome

(systemic inflammatory-

response-like syndrome)

Shock, haemodynamic changes, impaired

organ perfusion, disseminated intravascular

coagulation

Bioactive toxins; T

1 cytokines; acute-phase reactants

EMP1, erythrocyte membrane protein 1; GPI, glycosylphosphatidylinositol; IFN-γ, interferon-γ; IL-10, interleukin-10; RBC, red blood cell; T

, T helper; TNF, tumour-

necrosis factor.

ERYTHROPOIETIC

SUPPRESSION

The inhibition of normal

production of fresh red blood

cells in the bone marrow or

spleen. This occurs by various

mechanisms, including

inhibition of precursor-cell

responsiveness to

erythropoietin.

Divergent results might be a consequence of differences

in haemozoin preparations, because these preparations

are heterogeneous, containing uncharacterized bio-

active contaminants, such as non-covalently associated

phospholipids, hydroxylated fatty acids, carbohydrates

and glycolipids. Removal of non-covalently asso ciated

lipids from haemozoin preparations abolishes their

bioactivity

20,21

. However, a synthetic version of haemo-

zoin, β-haematin, has pro-inflammatory activity on

mouse monocytes and

macrophages

13,22

At high PRBC to target-cell ratios, PRBCs can

inhibit the maturation of DCs and reduce their

ability to stimulate T cells

23,24

. DC exhaustion could

result from antigen overload, but it has been sug-

gested, although not proven, that these activities

result from the binding of P. f a l c i p a r u m EMP1 to

CD36 and CD51

REFS 23,24. If this is the case, this

important molecule would also participate in the

negative regulation of the immune system, in con-

trast to PAMPs, which only induce activation of the

innate immune system.

Recognition of parasite molecules by innate immune

receptors. The various members of the mammalian

Toll-like receptor (TLR) family are important recep-

tors that are responsible for recognition of microbial

PAMPs. Mice that have a mutant version of TLR4,

which binds lipopolysaccharide, are responsive to GPI

from P. f a l c i p a r u m

and T. cruzi, indicating that this

glycolipid triggers a different receptor that results in

the expression of pro-inflammatory genes. GPI from

both pathogens can activate TLR2, and this requires

the crucial TLR adaptor protein MyD88 (myeloid

differentiation primary-response gene 88)

25,26

. However,

TLR2-deficient mice produced pro-inflammatory cyto-

kines when stimulated with live T. cruzi, and they were

almost as resistant to infection as wild-type mice

Evidence for the involvement of TLR mechanisms in

the immunoregulation and immunopathogenesis of

malaria is still limited. MyD88-deficient mice that are

infected with Plasmodium berghei have less liver injury

and produce less IL-12 but not IL-18 than wild-type

mice

. However, the liver injury in this experimental

system does not model the pathophysiological disease

processes that occur in humans. Haemozoin activates

mouse DCs through TLR9

REF. 13. TLR9 is expressed

by monocytes, macrophages, B cells and DCs in mice,

but it is restricted to B cells and plasmacytoid DCs

in humans

. So, TLR9 agonists (and presumably

haemozoin) do not activate monocytes or macrophages

in humans. TLR9 functions through a strictly MyD88-

dependent pathway. However, data indicate that

there is no significant difference in peak parasitaemia,

ERYTHROPOIETIC SUPPRESSION, interferon-γ (IFN-γ) produc-

tion and CM fatality rates between P. b e r g h e i -infected,

Myd88

–/–

mice and their P. berghei-infected, Myd88

+/–

litter-mates (L.S., unpublished observations). If this

observation is confirmed, then MyD88-independent

pathways would seem to dominate disease processes

in this model. Other lectin-like receptors might also

function as pattern-recognition receptors or modula-

tory receptors, including calcium-dependent C-type

lectins such as soluble mannose-binding lectin (MBL),

which is present in the plasma. MBL binds sugars

that are present at the surface of PRBCs but have yet

to be characterized

, and a low level of MBL in the

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Table 2 | Malaria products and their bioactivities

Parasite product Receptor and cell type Pathological and cellular effects

Plasmodium falciparum

EMP1-family members

ICAM1, VCAM1, CD36, thrombospondin,

E-selectin, chondroitin sulphate A,

hyaluronic acid and CD31 on endothelial

cells and trophoblast cells; CD36 on DCs

Binding directs parasite to the brain, placenta

and possibly other target organs; CD36

engagement proposed to suppress DC and

macrophage activation

GPI TLR2, TLR4 and/or possibly C-type

lectins on several cell types, including

DCs, macrophages, endothelial cells and

adipocytes; CD1d and Vα14–Vβ8 TCR

on NKT cells

Induces widespread expression of genes

encoding pro-inflammatory proteins (including

TNF, IL-1, IL-6, IL-12, iNOS, ICAM1, VCAM1);

activates NKT cells; induces T

1- or T

cytokine production

Haemozoin TLR9 on DCs Contradictory reports: both T

1- and T

cell activities; induces and inhibits DCs;

suppresses macrophages; induces IL-10

production; broadly immunosuppressive

Unknown ligands NKC-encoded receptors on NK and

NKT cells

Activates NK cells; induces IFN-γ production;

regulates balance of T

1 and T

2 cytokines

produced by NKT cells

Isopentenyl

pyrophosphate

γδ TCRs Activates γδ T cells; induces IFN-γ production

Protein antigens Diverse TCRs on CD4

and CD8

T cells Activates αβ T cells; induces T

1- or T

cytokine production

Unknown sugar(s) MBL in plasma Possible binding provides protection; low

levels of MBL are associated with disease

DC, dendritic cell; EMP1, erythrocyte membrane protein 1; E-selectin, endothelial-cell selectin; GPI, glycosylphosphatidylinositol;

ICAM1, intercellular adhesion molecule 1; IFN-γ, interferon-γ; IL, interleukin; iNOS, inducible nitric-oxide synthase; MBL, mannose-

binding lectin; NK, natural killer; NKC, natural killer complex; NKT, natural killer T; TCR, T-cell receptor; T

, T helper; TLR, Toll-like

receptor; TNF, tumour-necrosis factor; VCAM1, vascular cell-adhesion molecule 1.

plasma is associated with severe disease in humans

Nonetheless, lectin-like-receptor signalling and bio-

activity remain poorly defined. Further studies are

clearly required to dissect the role of purified, struc-

turally defined malaria PAMPs in the activation of

TLR-signalling pathways, both those that are MyD88

dependent and MyD88 independent, and to elucidate

the possible contributions of these pathways to relevant

pathophysiological processes.

‘Intermediate’ pathways between innate and adaptive

immunity. Many studies show that pro-inflammatory

1 cytokines are crucial determinants of malaria

disease states. In addition to acute-phase mono-

kines, the production of which is induced rapidly by

parasite toxins, IFN-γ levels can increase very early

during malaria, indicating that non-conventional lym-

phoid populations that can function with accelerated

kinetics account for this production. CD1d-restricted

natural killer T (NKT) cells are such an ‘intermedi-

ate’ arm between innate and adaptive immunity, and

these cells are particularly important in regulating the

downstream differentiation of CD4

T cells into T

and T

2 cells

. As well as having toxic bioactivity, GPI

is a natural glycolipid ligand for NKT cells

, together

with closely related myco bacterial phosphatidyl inositol

mannosides

. In mice with CM, NKT cells were shown

to be a crucial determinant of cytokine levels, the pro-

inflammatory cascade, pathogenesis and fatality

The CD1d–NKT-cell pathway also upregulates and

downregulates acute malarial splenomegaly in mice

and is an important determinant of B-cell responses

The CD1d–NKT-cell pathway either prevents or pro-

motes fatality, and it determines the differentiation of

immune cells, depending on which alleles of the natu-

ral killer complex (NKC), which is located on mouse

chromosome 6

REF. 37, are expressed. The loci in the

NKC are differentially expressed by natural killer (NK)

cells and NKT cells, and they control the production

of pro-inflammatory T

1 cytokines and counter-

regulatory T

2 cytokines by NKT cells

. NKC loci also

determine the level of malarial anaemia, the isotypes

of malaria-specific antibodies and the T

1/T

2 profile of

conventional T cells that is induced during infection

C57BL/6 mice, which are T

1-cell-response prone, are

susceptible to CM, whereas BALB/c mice, which have

a genetically determined T

2-cell bias, are resistant

and these different profiles reflect the substantial con-

tribution of polymorphic NKC loci

. Infection with

malaria-causing agents imparts NKC-dependent signals

to NKT cells that influence their differentiation into

cells that secrete T

1 or T

2 cytokines, but the spe-

cific receptor–ligand interactions that are involved in

this process are unknown. So, the NKC is a crucial

genetic determinant of malaria pathogenesis in mice,

with an important role in controlling NK- and NKT-

cell function. Not all NKC loci are involved, however,

because injecting certain NK-cell-specific monoclonal

antibodies does not affect pathogenesis in mice

Human NK cells also become activated early dur-

ing malaria

and are activated rapidly by parasites

in vitro

, which requires direct contact of PRBCs

with NK cells and results in IFN-γ production

However, the relevance of NK cells to human disease

remains unclear.

Progression to cerebral malaria

The histopathology of CM is associated with the

accumulation of mature PRBCs in cerebral micro-

vessels, through sequestration. This feature was

first described in 1894

REF. 43 and has since been

confirmed by numerous studies

. This has led to the

dominant theory of CM pathogenesis: that, because

PRBCs are sequestered in brain capillaries and post-

capillary venules, they induce flow perturbations

that eventually lead to obstruction and hypoxia of

the surrounding brain parenchyma

45,46

and to haem-

orrhages. Nevertheless, as early as 1944, there were

doubts about any causal relationship between CM and

PRBC sequestration

. Indeed, there is, for the most

part, no proof that PRBC sequestration is sufficient

to cause CM

and even less that it is a cause of death;

this issue has been debated previously

48,49

. Mature-

stage parasites are absent from the peripheral blood

of patients infected with P. falciparum. Clearly, seques-

tration of PRBCs in deep microvascular beds occurs

routinely in all of these patients, although only 1% of

these individuals develop CM. So, PRBC sequestra-

tion might not be sufficient to cause CM, but it might

be necessary. Subsequently, it has become apparent in

humans with CM, as well as in mouse models of CM,

that host cells, such as leukocytes or platelets, might

also be sequestered in brain microvessels, in addition

to PRBCs

50,51

. These host cells might be involved in

the pathogenesis of CM, either through local effects in

brain microvessels or through distant effects mediated

by the production of potentially deleterious mediators,

such as pro-inflammatory cytokines, which can be

detected in the circulation. These leukocytes, however,

show little evidence of endothelial extravasation and

therefore cannot be described as classic inflammatory

cells. The

TIMELINE shows how the study of mouse and

simian models, in vitro assays and human infections

has contributed over time to the elucidation of the

complex cascade that controls CM pathogenesis.

Accumulation of intravascular infiltrates. The intra-

vascular accumulation of monocytes in the brain has

long been recognized in mice

52–54

and humans

51,55–60

with CM. Sequestered monocytes and macrophages are

more abundant in paediatric patients with CM than in

those with SMA or non-malarial encephalopathy

. This

feature has largely been ignored as a possible contributor

to the pathogenic process in CM in humans.

In CM, some cells of the monocyte–macrophage

lineage within the microvasculature show charac-

teristics that are normally associated with tissue

macrophages (including an increased size and the

presence of phagocytosed material, vacuoles, a ruffled

plasma membrane and pseudopods). For this reason,

we refer to these cells as intravascular macrophages.

Therefore, CM is a rare situation in which monocytes

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differentiate into macrophages in the intravascular

compartment and not in the tissues. Several modi-

fications of monocyte–macrophage phenotype and

functions are known to occur in severe malaria. For

example, coagulation factors secreted by monocytes,

in addition to their role in blood-clot formation,

might contribute to the sequestration of cells in brain

microvessels of patients with CM, at least in children.

Tissue factor can itself function as an adhesion mol-

ecule, and it has coagulation-independent roles in cell

adhesion and migration. Furthermore, upregulation

of tissue-factor expression has a central role in driv-

ing a thrombosis–inflammation circuit. Coagulant

mediators (such as factor VIIa and factor Xa) and the

end-product fibrin are also pro-inflammatory, elicit-

ing the expression of TNF and other cytokines, as well

as chemokines and adhesion molecules, and this has

recently been reviewed in

REFS 50,51.

The accumulation of leukocytes in the brains

of patients with CM is evidence of a consider-

able chemokine cascade, which has been shown in

experimental studies, including DNA-microarray

analyses of mouse disease

61,62

. The expression of

monocyte-secreted cytokines and chemokines, such

as TNF, CXC-chemokine ligand 10 (CXCL10; also

known as IP10), CC-chemokine ligand 2 (CCL2;

also known as MCP1) and CCL5 (also known as

RANTES), varies with mouse genotype and corre-

lates with resistance versus susceptibility to disease

Neutrophils also contribute to the brain lesions

in mice and are an important source of cytokines

(including the p40 subunit of IL-12, IL-18, IFN-γ

and TNF) and chemokines (including CCL3 (also

known as MIP1α), CXCL9 (also known as MIG) and

CXCL10) that participate in pathogenesis. Indeed,

depletion of neutrophils early in malaria prevents

the development of CM in mice, downregulates the

expression of T

1 cytokines in the brain, and mark-

edly decreases the sequestration of monocytes and

the incidence of microhaemorrhages in the brain

Although the contribution of neutrophils to CM in

humans is unknown, the neutrophil-specific acti-

vation marker and recruitment agent lipocalin was

found at a higher concentration in plasma from

patients with severe malaria

, and transcription of

the gene encoding lipocalin is upregulated in the

brain during cerebral disease in mice

Evidence for a pathogenic role of platelets,

both in CM in mice and in in vitro models of CM

in humans, has been summarized elsewhere

. As

illustrated in

FIG. 2, there are several possible ways

through which platelets could affect endothelial-

cell function and viability, and promote leukocyte

adhesion. First, platelets, together with other cell

types, can modulate the expression of adhesion

molecules, such as ICAM1, and the production of

cytokines, such as IL-6, by endothelial cells

, through

the release of IL-1. Second, platelet-derived micro-

particles modulate endothelial-cell metabolism, by

regulating the production of cyclooxygenase-2 and

prostaglandins

, and increase the adhesiveness of

the endothelial-cell–leukocyte–platelet interaction,

Timeline | Contribution of animal models and human-based assays to our understanding of cerebral malaria

PRBC sequestration

T-cell dependency

of disease

PRBC

sequestration

Lactate

detected in

cerebrospinal

fluid

Brain lesion

observed

Leukocyte

sequestration

CD4-specific

antibody

protects

TNF-

specific

antibody

protects

CD36 is a receptor for

PRBC sequestration

TNF detected

in plasma

LFA1-specific antibody protects

ICAM1 is a

receptor for PRBC

sequestration

Blood–

brain barrier

alterations

observed

IFN- -specific

antibody

protects

GPI is

a toxin

Platelet-specific antibody protects

Role for

neutrophils

P. falciparum EMP1

is a ligand for PRBC

sequestration

Axonal injury

observed

Presentation of GPI by CD1d

Kynurenine

metabolism in

cerebrospinal

fluid

Platelet

sequestration

Axonal injury

observed

Microparticles

detected in

plasma

Role for ATP-

binding cassette

transporter

PRBC sequestration

P. falciparum EMP1

first identified

VCAM1 is a receptor

for PRBC

sequestration

Nitric oxide

protects

Role for

TNFR2

Role for

and V 8

T cells

Role for

CD8

T cells

Role for lymphotoxin-α

Role for

NKT cells

Role for

CCR5

1900 1980 1982 1984 1985 1986 1987 1989 1991 1992 1993 1994 1997 1998 1999 2002 2003 2004 2005

Duffy-binding-like

domains are ligands

for PRBC binding

Role for

endothelial-

cell-

expressed

P-selectin

Research on cerebral malaria has been carried out in mouse in vivo infection models (orange), simian in vivo infection models (green), in vitro models of human infection (yellow) and

in vitro assays involving human tissues (blue). CCR5, CC-chemokine receptor 5; EMP1, erythrocyte membrane protein 1 of P. falciparum; E-selectin, endothelial-cell selectin; GPI,

glycosylphosphatidylinositol; ICAM1, intercellular adhesion molecule 1; IFN-γ, interferon-γ; LFA1, lymphocyte function-associated antigen 1; NKT, natural killer T; P. falciparum,

Plasmodium falciparum; PRBC, parasitized red blood cell; P-selectin, platelet selectin; TNF, tumour-necrosis factor; TNFR2, TNF receptor 2; VCAM1, vascular cell-adhesion molecule 1.

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NUDE MICE

Mice with a mutation that

causes both hairlessness and

defective formation of the

thymus, which results in

a lack of mature T cells.

by directly upregulating the expression of adhesion

molecules and integrins such as ICAM1 and macro-

phage receptor 1 (

MAC1; CD11b–CD18), both by the

endothelium and by adherent monocytes and other

leukocytes

. Consistent with this, microparticles have

been shown to be crucial for the development of CM

in mice

, and the level of microparticles is markedly

increased in the plasma of children with CM

. Third,

the adhesion of activated platelets to the endothelium

(which is an early event in inflammation and possibly

occurs through CD40-ligand–CD40 interactions)

can indirectly mediate leukocyte–endothelial-cell

adhesion, by providing additional receptors, such as

fibrinogen and

ICAM2 REF. 71, which are ligands for

leukocyte MAC1 and lymphocyte function-associated

antigen 1 (

LFA1; CD11a–CD18), respectively. In

addition, platelet selectin (

P-selectin) is released

from secretory granules of activated platelets and

can be expressed at their surface, and leukocytes

that express

P-selectin glycoprotein ligand 1 can then

adhere to this surface P-selectin

. Last, platelets can

also modulate the sequestration of normal RBCs and

of PRBCs, by surface expression of CD36, which

binds P. f a l c i p a r u m EMP1 at the PRBC surface, and

they can directly modulate cytokine production by

circulating leukocytes and the endothelium, as has

been shown in mouse models of CM

A key unresolved question is how much of the

accumulation of parasites in cerebral vessels is associ-

ated with adhesion mediated by classic receptor–ligand

interactions and how much is associated with non-

specific deposition of activated platelets, deposition

of fibrin and the presence of other markers of the host

inflammatory response. Evidence from P-selectin- or

ICAM1-deficient mice indicates that most platelet

binding is likely to be ligand mediated (not non-

specific), because platelet binding is undetectable

by intravital microscopy in these mutant mice

Nonetheless, the relative importance of the numerous

effects of platelets in the pathogenesis of CM remains

to be further elucidated

Role of T cells. In mice, CM has been known to be a

T-cell-dependent disease for two decades.

NUDE MICE

and mice that are deficient in the αβ

-TCR are resistant

to disease. In addition, in vivo depletion using specific

monoclonal antibodies showed that CD4

T cells are

required for pathogenesis

. However, MHC-class-II-

deficient mice, which lack conventional CD4

T cells,

still develop CM

, probably by retaining CD1d-

restricted, CD4

NKT cells that have a crucial role in

disease

. Subsequently, γδ T cells have been shown to

participate in CM, because although mice that are defi-

cient in the γδ

-TCR are susceptible to CM, depletion

Figure 2 | Mechanisms of platelet–endothelial-cell interactions in cerebral malaria. Platelets, after activation by tumour-

necrosis factor, can markedly alter the functions of brain endothelial cells, either directly, by binding to the endothelium, or

indirectly, by releasing molecules from their secretory granules. For example, platelets release interleukin-1 (IL-1), which increases

the expression of adhesion molecules (such as intercellular adhesion molecule 1, ICAM1) and the production of cytokines (such

as IL-6) by endothelial cells. Platelet-derived microparticles also alter endothelial-cell metabolism by regulating the production of

cyclooxygenase-2 and prostaglandins, which might affect endothelium permeability, electrical resistance and apoptosis. One

of the earliest events in inflammation or tissue injury is the adhesion of activated platelets to the endothelium (possibly through

CD40 ligand (CD40L)–CD40 interactions). Importantly, in cerebral malaria, this provides additional receptors at the endothelial-cell

surface for the adhesion of leukocytes, which bind platelet surface molecules such as platelet selectin (P-selectin) and CD40L,

through P-selectin glycoprotein ligand 1 (PSGL1) and CD40, respectively. Furthermore, platelet-derived microparticles can

increase the adhesiveness of endothelial cells to leukocytes and the adhesiveness of the platelets themselves to the endothelium.

Finally, activated platelets can indirectly alter endothelial cells through the complex effects of their granule-derived mediators

on leukocytes, including platelet-derived growth factor (PDGF), 12-hydroxyeicosatetraenoic acid (12-HETE), CXC-chemokine

ligand 4 (CXCL4; also known as PF4), β-thromboglobulin (β-TG) and transforming growth factor-β (TGF-β). The exact

mechanisms that are involved have yet to be defined. EMP1, erythrocyte membrane protein 1 of Plasmodium falciparum;

LFA1, lymphocyte function-associated antigen 1; MAC1, macrophage receptor 1; PRBC, parasitized red blood cell.

ICAM1

PSGL1

Brain

endothelial cell

Platelet

Microparticle

Leukocyte

CD40

MAC1

ICAM2

Fibrinogen

CD40L

IL-1

Release of PDGF, 12-HETE,

CXCL4, β-TG and TGF-β

CD36

EMP1

PRBC

LFA1

P-selectin

Effects on endothelium

↑ Adhesiveness for leukocytes and PRBCs

↑ Permeability

↓ Electrical resistance

↑ Apoptosis

728

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REVIEWS

of γδ T cells from wild-type mice (using specific anti-

bodies) confers resistance to CM. γδ-TCR-deficient ani-

mals therefore seem to develop a functional redundancy

that is absent in conditions of normal immunological

ontogeny

. In humans, there are few data that address

T-cell involvement in CM.

IFN-γ seems to be the most important T-cell-

secreted cytokine. In vivo neutralization of IFN-γ in

mice infected with P. berghei strain ANKA prevented

TNF overproduction and CM development

. The

central role of IFN-γ in the pathogenesis of CM was

confirmed by experiments in mice that were defi-

cient in IFN-γ or the IFN-γ receptor, which are resis-

tant to the development of experimentally induced

. The cell-surface antigen CTLA4 (cytotoxic

T-lymphocyte antigen 4; CD152) negatively regu-

lates T cells and therefore might downregulate

T-cell responses and prevent immunopathology. As

expected, blockade of CTLA4 exacerbated CM in mice

which highlights the contribution of T cells to disease.

CD8

T cells might also be effectors in patho-

genesis

39,80,81

. In mice, the number of cytotoxic CD8

T cells infiltrating the brain during CM is increased,

and these cells contribute to permeability changes

of the mouse blood–brain barrier through perforin-

dependent mechanisms

82,83

. CM is also associated with

an increase in the number of peripheral CD8

T cells

that have TCRs using the Vβ8.1 or Vβ8.2 segments

and disease was reduced when mice were treated with

antibodies that specifically neutralize these T cells.

Because of the hypothesis that CD8

T cells contribute

to the pathology of CM

, the role of CC-chemokine

receptor 2 (CCR2), which is expressed by CD8

T cells,

was evaluated

. Mice that were deficient in CCR2

remained susceptible to CM, indicating that this

receptor is not directly involved in cerebral pathol-

ogy. However, the number of CD8

CCR5

T cells

in the brain increased after infection with P. berghei

strain ANKA, and CCR5-deficient mice were partially

protected against the neurological syndrome

. CD8

T cells sequestered in the brain were proposed to be

responsible for the neurological syndrome and for

death, but there has been no report that directly shows

the presence of these cells, using histopathology, at the

site of neurovascular lesions. Direct contact has been

shown in vitro between brain endothelial cells and

activated T cells, and this contact might also be cru-

cial in cerebral pathogenesis

. CD8

T cells also have

a role in circulatory shock and in respiratory distress

in mice that are infected with P. berghei

Microvascular obstruction: what is the sequence

of events? In conclusion, histopathological studies

have uncovered the presence of PRBCs, platelets and

leuko cytes, each of which might contribute to dis-

ease, in the cerebral vascular lumen in humans and

mice with CM

(FIG. 3). What cannot be deduced from

such observations is the order in which the cells and

platelets are sequestered in a vessel and the nature

of the ensuing interactions. There are three possi-

bilities: namely, that PRBCs bind brain endothelial

cells first, followed by leukocytes or platelets; that

leukocytes bind first; or that platelets make the initial

interaction. This might vary in different areas of the

brain and over time, thereby leading to a mosaic of

arrested cells. So, conclusions drawn from histology,

even in post-mortem analyses of multiple sites, might

be influenced by sampling variables. In addition, the

transient nature of the binding events, the temporal

pattern of expression of adhesion molecules, and

competition among PRBCs and leukocytes for bind-

ing sites is likely to determine the sequence of adhe-

sive events. For example, P-selectin and E-selectin

are expressed transiently, which probably influences

the binding of circulating cells. Endothelial-cell,

leuko cyte and platelet adhesion molecules are likely

to participate in cell–cell interactions in this dynamic

environment. After cells have accumulated, they pro-

duce chemotactic, inflammatory and toxic mediators

that further contribute to the pathogenesis, and this

can lead to positive-feedback cycles

(FIG. 3).

Judgement must remain reserved, however, on the

contribution of each factor in different age groups, in

different transmission settings and across the spec-

trum of disease. What is cause and what is effect in

this complex picture? The difficulty of ascribing func-

tion applies particularly to post-mortem histological

analyses of human cerebral disease states, because

processes that are observed to be associated with

disease might be corrective attempts that have been

made by the host to control infection or to downregu-

late pathogenesis, or might be pathogenically neutral.

A recent histological study points to the possibility of

a disease spectrum in paediatric patients with CM

There are three defined disease categories (CM1,

CM2 and CM3), and they occurred in 15%, 56% and

29% of clinically defined cases, respectively. Patients

with CM1 have only PRBC sequestration, whereas

patients with CM2 have PRBC sequestration plus

other intra- and perivascular pathology, including

immune-cell infiltrates. Patients with CM3 fulfilled

the complete World Health Organization clinical

criteria for CM, including unrousable coma associ-

ated with infection, but they died of non-malarial

causes. So, what seems to be a defined syndrome

appears heterogeneous with respect to the under-

lying pathogenesis

. Key histological studies have

been undertaken in South-East Asian adults. These

implicate a role mainly for PRBC sequestration in

CM, and it seems probable that there are substantial

differences between CM in adults and in children

(studied in Africa), with intravascular leukocyte

accumulation being more pronounced in children.

Fortunately, animal models allow hypotheses to be

tested by experimental intervention, and CM in

rodents seems to mirror the human disease with

reasonable accuracy, including presenting a disease

spectrum that depends on host genetics. Because

disease progression in animal models evidently

requires several sequential steps, multiple points of

intervention might also be possible in the treatment

of humans with CM.

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Immunology of placental malaria

In areas with high rates of transmission of malaria,

women have considerable immunity to malaria by the

time they reach child-bearing age. However, during

their first or second pregnancy, they are susceptible

to placental malaria, and this becomes less common

with subsequent pregnancies. CSA and hyaluronic acid

are receptors that are expressed by endothelial cells,

preferentially in the placenta. PRBCs that display

P. falciparum EMP1 proteins that bind these recep-

tors can be sequestered, allowing parasite maturation

on the placental endothelium

and thereby validating

the link between an adhesive phenotype and organ-

specific disease

. As is the case for CM in mice

52,54,91

and humans

51,55,60

, severe placental malaria (which

results in low birth weight) is associated with sub-

stantial macrophage infiltration into the placenta

and high chemokine expression

92,93

, indicating that

adhesive phenotype and chemokine-driven cellular

infiltration are key determinants of organ-specific dis-

ease syndromes. Interestingly, women with placental

malaria remain afebrile, indicating that the condition is

mainly a local pathology, with some aspects of acquired

clinical immunity remaining unaffected by placental

parasitization.

Immunology of severe malarial anaemia

SMA is the most serious and most common pernicious

complication of malaria, and it might be the leading

cause of deaths from malaria worldwide. Compared

with the florid, acute signs of CM, SMA can be chronic

or silent and therefore is less well studied. However,

that peaks of SMA incidence coincide with peaks of

CM incidence during high transmission seasons in

endemic areas indicates that SMA might sometimes

be an acute syndrome, notwithstanding that, for some

cases, this might be the point at which a chronic process

flares up.

Many cross-sectional studies report a lack

of correlation between parasite densities and severity of

anaemia, although longitudinal studies indicate that

duration of infection might be a better predictor of risk

of disease

. There is a misconception, however, that

SMA arises simply from the destruction of infected

RBCs, and over-reliance on inappropriate models

might contribute to this view

BOX 1. Many animals

with acute infections develop severe haemolytic anae-

mia, through invasion and rupture of RBCs as a con-

sequence of excessive parasite burdens of a magnitude

that is rare in humans with malaria. In humans, SMA

is typically associated with parasite burdens that are

considerably lower than those required for the marked,

direct destruction of RBCs

, with ∼12 uninfected RBCs

lost for every PRBC

95,96

. Because of the limitations of

hyperparasitaemic experimental infections, there has

been increased interest in Aotus spp. monkeys either

immunized with experimental vaccines consisting

of antigens from blood-stage parasites or rendered

semi-immune by previous exposure to infection with

P. f a l c i p a r u m , as these animals develop marked SMA

despite having very low parasite burdens

97,98

. Before the

advent of antibiotics, the high fever induced by malaria

was used to treat neurosyphilis; the low parasite bur-

dens, and the kinetics and magnitude of SMA, in

semi-immune simian models mirror the anaemia that

developed in the numerous untreated humans who

had malaria that was induced for the management of

neurosyphilis

95,99,100

. Therefore, because direct destruc-

tion of RBCs by parasitization seems to be a relatively

Figure 3 | Schematic representation of events that are likely to lead to severe malarial

disease, particularly in the brain. First, parasitized red blood cells (PRBCs) adhere to

receptors expressed by brain microvascular endothelial cells, such as intercellular adhesion

molecule 1 (ICAM1), through surface expression of Plasmodium falciparum erythrocyte

membrane protein 1 (EMP1). When merozoites are released from PRBCs ∼4 hours later, parasite

glycosylphosphatidylinositol (GPI), which is either released into the blood or present in

parasite membranes, functions as a pathogen-associated molecular pattern and toxin, thereby

inducing an inflammatory response. A local acute-phase response then occurs, which involves

activation of the endothelium and local production of cytokines and chemokines, and this

results in upregulation of expression of cell-adhesion molecules by endothelial cells. Within

the next ∼24 hours, this cycle is perpetuated and exacerbated, owing to increasing parasite

numbers and further binding of PRBCs to endothelial cells that have upregulated expression

of cell-adhesion molecules. GPI can also function as a ligand for CD1d-restricted natural killer

T (NKT) cells, leading to their activation. Activated NKT cells can regulate the differentiation of

CD4

T cells into T helper 1 (T

1) or T

2 cells, depending on which natural-killer-complex loci

are expressed, so activation and involvement of CD4

T cells occurs. In addition, chemokines

recruit monocytes and activate neutrophils (although neutrophils are not known to infiltrate

brain microvessels in humans or mice with cerebral malaria). Recruited monocytes can then

differentiate into macrophages and become arrested in brain microvessels. Macrophages

can also be activated by GPI, a process that is amplified by interferon-γ. Local activated

macrophages produce more chemokines, which are released systemically, thereby amplifying

infiltration of cells, sequestration of PRBCs and release of microparticles (which are probably

of endothelial-cell origin). After several more cycles, γδ T cells and CD8

T cells might become

involved, releasing more chemokines and cytokines both systemically and locally and possibly

inducing perforin-mediated lesions in the endothelium. Together with locally arrested

macrophages, platelets are sequestered and participate in altering endothelial-cell functions.

More microparticles of platelet, endothelial-cell and monocyte origin are released, which leads

to the dissemination of pro-inflammatory and pro-coagulant effects. Finally, damage to the

endothelium, with possible perivascular haemorrhage, axonal injury, and neurotransmitter

and metabolic changes, can ensue. The overall disease spectrum in humans might depend

on whether all of these processes occur or only some of them.

Release

of GPI

Local expression

of cell-adhesion

molecules increases

Cytokine and chemokine

cascades occur

First wave of

microparticles

Second wave

of microparticles

Local production

of cytokines and

chemokines increases

Systemic cytokine

levels increase

Systemic cytokine

levels increase

Local cytokines

levels increase

Endothelial-cell

functions alter

Bleeding,

hypoxia(?),

parenchymal

and axonal

damage

Endothelial cell

Parasitized red

blood cell

Platelet

Monocyte

NKT cell

Neutrophil

CD4

T cell

CD8

T cell

Macrophage

EMP1

γδ

T cell

730

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REVIEWS

RETICULOENDOTHELIAL

SYSTEM

The general phagocytic system

of the host. It is responsible for

removal and destruction of

foreign material and senescent

or dead host cells, such as red

blood cells.

minor contributory mechanism

101

, SMA is thought to

arise mainly from two processes: increased destruction

of non-parasitized RBCs, and decreased production of

RBCs (also known as erythropoietic suppression).

Destruction of uninfected RBCs. In SMA in humans,

accelerated RBC turnover is proposed to result from

changes to the surface or structure of uninfected RBCs

that target them for destruction

102

. The following have

been detected at relatively high frequencies in indi-

viduals with SMA

101

: oxidation, phosphatidylserine

externalization and reduced deformability of RBCs;

complement binding by RBCs; complement

regulatory

deficiencies; and autoantibodies, immune complexes

and IgG specific for non-specifically adsorbed para-

site antigen at the surface of RBCs. These mechanisms

are proposed to target RBCs for destruction by intra-

vascular haemolysis or for clearance mediated by the

RETICULOENDOTHELIAL SYSTEM (RES). However, human

SMA is not associated with the typical signs of intra-

vascular haemolysis, such as secretion of haemo globin

in the urine. By contrast, homeostatic RES clearance

of RBCs is mainly mediated by macrophages in the

splenic red pulp, and recruitment to the spleen or

activation of these macrophages might accelerate

the process

103,104

. SMA is associated with high serum

levels of neopterin, a marker of macrophage activa-

tion, the expression of which is induced by IFN-γ in

particular

103

. Phagocytosis of uninfected RBCs has

been documented during human infections

105,106

, and

upregulation of RES activity is implicated in malarial

thrombocytopaenia

107

. Anaemia often persists after

clearance of parasitaemia, and haemo globin normal-

ization shows delayed kinetics compared with condi-

tions that have similar blood loss, such as trauma

It is possible that clearance by the RES that involves

high RBC turnover extends beyond the period of

parasitaemia. In untreated malaria used to control

neurosyphilis, the rapid onset of severe anaemia was

temporally linked to the appearance of a distended

spleen, as was subsequently observed in children with

malarial anaemia

. These observations are consistent

with hyperactivation of the RES and with the magni-

tude of RBC destruction during SMA. This has led to

the proposal that SMA has an inflammatory aetiol-

ogy

103,104

. Counter-regulatory T

2 cytokines such as

IL-4

REF. 103 or IL-10 REFS 108,109 might therefore

protect against disease. So, SMA might be regulated by

diverse factors that control hypersplenism and splenic

macrophage recruitment and activation: for example,

host CD4

T cells, cytokines and chemokines (such

as CCL2, CCL3 and CCL4 (also known as MIP1β))

and parasite products (such as haemozoin and GPI).

SMA might therefore be precipitated by the rise of

parasitaemia above a crucial threshold, as has been

observed for experimental infections of simians

97,98

and humans

95,99,100

Nevertheless, it might be the immune response of the

host to this parasite biomass that is the main cause of

anaemia, rather than simply the direct destruction

of RBCs by parasitization. In broad terms of immuno-

regulation, SMA might therefore have similarities

to other malaria syndromes. Adoptive transfer of

parasite-specific, CD4

1-cell lines promotes anaemia

after infection with P. berghei

110

, showing that antigen-

specific T cells can contribute to the severity of disease.

A plausible mechanism lies in the ability of T cells to

upregulate macrophage-mediated RBC clearance.

Because SMA is marked in Aotus

spp. monkeys that

have been immunized with P. f a l c i p a r u m antigens

97,98

blood-stage vaccines might not necessarily protect

against this condition, although they might suppress

parasitaemia. Whether T-cell priming by these experi-

mental vaccines promotes the development of SMA is

not clear. Such preclinical models might prove useful

for further assessing the efficacy of vaccines in the

prevention or promotion of this disease state.

Decreased production of RBCs. Normal homeostasis of

RBC numbers is maintained by balancing destruction

of old RBCs by the RES with production of new RBCs

through erythropoiesis. Under the influence of factors

such as erythropoietin, haematopoietic stem cells in

the bone marrow or spleen multiply and differentiate

to produce the youngest fully functional RBCs, which

Box 1 | Experimental models of malaria

The study of malaria is aided by the availability of a wide range of experimental

models. Various species of the Plasmodium genus — such as Plasmodium chabaudi,

Plasmodium berghei, Plasmodium yoelii and Plasmodium vinckei — naturally infect

rodents. Adapted to infect laboratory rats and inbred mouse strains, they have

proven enormously useful in the development of drugs and vaccines, and in basic

studies of pathogenesis, immunology, and the genetics of susceptibility or resistance

to infection and disease. Other models use parasites that cause malaria in simians —

such as Plasmodium knowlesi and Plasmodium cynomolgi — or parasites of humans

that are adapted to simian hosts (such as Aotus spp. monkeys). Although no two

host–parasite combinations are identical in all features of the relationship, some

models are better than others at recapitulating the main features of pathology or

the immune responses that occur in malaria in humans. There is great diversity

of responses and disease outcomes in human populations, so experimental models

that use inbred hosts might reflect only a section of the natural spectrum of disease

in humans: varying both host and parasite genetics uncovers a diversified disease

spectrum in these models.

Malaria in mice

Early during infection, P. be r g h e i strain ANKA is a good model of cerebral malaria

(CM), and processes identified using this model have been subsequently validated

in humans. Inbred mouse strains differ markedly in their susceptibility, showing the

importance of host genetic variation in immunopathogenesis

133

. Similarly, different

strains of P. be r g h e i (K173 versus ANKA) differ in some aspects of pathogenesis,

indicating the influence of parasite genetic variation in induced pathology.

Infection with P. yoelii strain 17XL (a lethal strain) induces CM that is associated

with the sequestration of parasitized red blood cells, and it has been used together with

P. yoelii strain 17XNL (a non-lethal strain) to study experimental vaccine-induced

immune responses.

P. chabaudi chabaudi strain AS causes a non-lethal infection in resistant mouse

strains and a lethal infection in susceptible mouse strains. Lethality, however, results

from haemolysis that is secondary to hyperparasitaemia, which might not be relevant

to the human disease processes. This Plasmodium strain has been used to study

experimental vaccines and immunological processes that control hyperparasitaemia.

Infections with P. chabaudi adami are self-resolving, non-pathogenic and non-lethal.

P. vinckei vinckei causes an aggressive, overwhelming hyperparasitaemia.

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are known as reticulocytes and are the earliest cell in

this pathway to be released into circulation. The num-

ber of reticulocytes in the blood directly reflects recent

erythropoietic activity. Reticulocyte levels are often

decreased during acute infection with P. falciparum,

indicating that another mechanism might contribute

to SMA — erythropoietic suppression

111

. Animal mod-

els seem to accurately recapitulate this phenomenon.

Infection with Plasmodium chabaudi or P. b e r g h e i

results in decreased proliferation, differentiation and

maturation of erythroid precursors

112,113

, and reticulo-

cyte levels are decreased early in infection, with DNA-

microarray analysis of splenocytes and bone-marrow

cells uncovering reduced transcription of at least 25

erythroid-specific loci

. It has been widely proposed that

the TNF and IFN-γ cytokine cascade that is associated

with the immediate early acute phase of infection medi-

ates erythropoietic suppression in mice and humans,

by decreasing the responsiveness of erythroid precur-

sors to erythropoietin

114,115

, but this remains unproven.

Anaemia in response to acute infection with P. berghei

strain ANKA is controlled, in part, by genes encoded

by the polymorphic NKC loci that are expressed by

NK and NKT cells, which control cytokine levels and

immune-cell differentiation

. Crude P. berghei strain

ANKA and P. chab au d i parasite lysates also induce

erythropoietic suppression in vivo

116

, reflecting the

bioactivity of parasite products (possibly haemozoin or

GPI), which either directly affect erythroid precursors

or indirectly affect them by influencing macrophages

(to secrete TNF

) or NKT cells

After loss of RBCs in conditions such as trauma,

anaemia is normally compensated for by physio-

logical erythropoiesis. However, SMA could be

exacerbated if erythropoietic suppression were to

prevent adequate reticulocyte compensation during

continuing clearance of RBCs. Further investigation

of the multiple host and parasite factors that might

influence these two contributory mechanisms of

SMA in vivo is required. Specifically, the impact

of vaccination on SMA-related end-points should be

examined in more detail in experimental models.

Clinical immunity to malaria

Epidemiological studies show that, after the initial

period in which children are susceptible to severe

malaria, protective immunity that is acquired to malaria

develops in three sequential phases: first, immunity to

life-threatening disease; second, immunity to symp-

tomatic infection; and only then, third, partial immu-

nity to parasitization. In 1899, Robert Koch observed

that immunity to disease precedes the ability to control

parasite densities, as others subsequently observed

117–119

proposing that it reflects the primary acquisition of

antitoxic immunity. The data from the treatment

of neurosyphilis with malaria shows evidence for anti-

toxic immunity

120

. At the population level, immunity

to severe malaria seems to be acquired after only one or

two infections

121

, although many children with severe

disease have a previous history of multiple mild bouts

of malaria.

Several studies report associations between levels of

antibodies that are specific for various parasite antigens

and reduced risk of infection

122

, but this is not clearly

established for disease states such as CM or SMA.

So, there is no single correlate of clinical immunity,

and those described do not account for the overall

variation in susceptibility in a population

123

. However,

antibodies specific for the parasite glycolipid GPI have

been found to be negatively associated with the risk

of developing SMA

or CM

124

and with acute febrile

episodes

125

, although a cross-sectional study found no

association with tolerance for parasitaemia

126

, which

might reflect the developmentally compromised acqui-

sition of carbohydrate-specific antibodies in infants.

Although clinical immunity might result from adaptive

immune responses to GPI, other explanations include

the acquisition of physiological non-responsiveness to

malaria toxins (which is analogous to tachyphylaxis,

the process of downregulation of lipopolysaccharide-

responsive signalling pathways following exposure

to the agonist). However, this mechanism is unlikely to

operate over long time-scales, whereas clinical immu-

nity seems to be relatively robust. A further possibility

is that disease susceptibility or resistance is regulated

to a considerable extent by the T

1/T

2-cytokine

profile of the NK- and NKT-cell arm of the immune

system (which is intermediate between the innate and

adaptive immune systems)

32,33,35–37

or of conventional

CD4

T cells and that clinical immunity is associated

with a switch away from the default, T

1-cell-biased

responses to T

2-cell-biased responses, which prevents

severe disease but controls parasite densities only after

an appropriately diverse antibody repertoire is gener-

ated. Establishing whether clinical immunity results

from adaptive immune responses to bioactive parasite

products, physiological desensitization to malaria tox-

ins, regulation of the balance of T

1 and T

2 cytokines,

or a combination of mechanisms is an important issue

for future research because such considerations should

inform vaccine development.

Implications for vaccines

Vaccines against malaria should aim to reduce mor-

bidity and mortality. Traditional approaches seek to

achieve this objective by reducing parasite burdens.

In support of this, a recent clinical trial shows that

reducing the infective inoculum by administration of a

sporozoite-specific vaccine reduces the rates of dis-

ease

127

. Nonetheless, reducing the replication of blood-

stage parasites, although likely to confer protection, will

not necessarily reduce morbidity or mortality, because

host immune responses, which can be non-linear with

respect to parasite densities, are important determi-

nants of these events. Despite the clinical objective of

vaccination, there has been no systematic attempt to

assess the impact of experimental vaccines in preclini-

cal models that have appropriate disease end-points.

Preclinical models that have been used so far include

infection of naive mice with P. c h a b a u d i and infec-

tion of naive Aotus

spp. monkeys with P. f a l c i p a r u m ,

and these infections result in high rates of parasite

732

SEPTEMBER 2005

VOLUME 5 www.nature.com/reviews/immunol

REVIEWS

1. Baird, J. K., Masbar, S., Basri, H., Tirtokusumo, S. &

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replication but not in clinically relevant syndromes.

As mentioned earlier, SMA is marked in Aotus

spp.

monkeys that have been immunized with P. f a l c i p a r u m

antigens

97,98

, so blood-stage vaccines might not neces-

sarily protect against this condition, although they

might suppress parasitaemia. Whether experimental

vaccines can induce immune responses that promote

SMA is not clear.

The use of vaccines against the disease aims to

reduce morbidity and mortality directly, by immuniz-

ing individuals with parasite products that contribute

to host pathology

128

. For example, vaccines that are

designed to prevent malaria during pregnancy target

the domains of P. f a l c i p ar u m EMP1 that bind placental

receptors

129

(that is, CSA and hyaluronic acid). Other

organ-specific disease processes could be targeted if a

restricted set of P. fal c i p a r u m EMP1 molecules were

found to be important

129

, the inherent diversity of these

targets being a potential barrier to these strategies.

Chemical synthesis of the glycan group of Plasmodium

spp. GPI, which is highly conserved and non-toxic, has

recently provided a means to test the hypothesis that

this molecule is causally involved in the pathogenesis of

P. berghei infection

130

. Vaccination with the GPI glycan

protects against blood acidosis, pulmonary oedema,

vascular occlusion by macrophages, and cerebral fatali-

ties in a rodent model of severe malaria induced by

P. b e r g h e i infection

130

. These findings show the efficacy

of a prototype antitoxic vaccine, and they prove that

GPI is an essential parasite product in the pathogenesis

of systemic disease and in the lethal cerebral syndrome

in this model.

It has been argued that antitoxic vaccines might

exacerbate disease by inhibiting the acute-phase

responses that limit parasite replication

131

. Indeed, it

has been proposed that the function of malaria toxins

is to elicit host responses that limit infection densities

by killing parasites

131

. However, ‘suicide’ explanations

of biological function in unicellular organisms are not

easily reconciled with Darwinian logic, it being difficult

to envisage why parasites would produce a molecule to

cause their own demise. Furthermore, strong acute-

phase immune responses are seen in association with

high parasite densities in sick children, and marked

inflammatory cascades do not necessarily reduce

parasite burdens in humans or in experimental models.

Clearly, this is an important area for further investiga-

tion. Nonetheless, these speculations help to highlight

that the impact of the current blood-stage vaccines on

malaria pathogenesis remains to be determined, even

in preclinical models.

Conclusions

Because they are blood-borne, malaria-causing para-

sites have access to multiple organs, including the

bone marrow, spleen, brain, lungs and placenta. The

binding of P. falciparum EMP1 to diverse endothelial-

cell-expressed receptors concentrates parasites

in certain sites. Parasite toxins, such as GPI and

haemo zoin, induce acute-phase immune responses,

with local activation of monocytes and the vascular

endothelium. GPI also contributes to early IFN-γ and

counter-regulatory IL-4 production, by functioning as

a ligand for the CD1d-restricted NKT-cell arm of the

immune system. The overall balance of T

1 and T

cytokines at this stage of the cascade is determined

to a considerable extent by the polymorphic loci in

the NKC. Chemokine cascades recruit intravascular

macrophages and inflammatory cells to diverse target

organs, with subsequent deposition of platelets and

fibrin. Various lymphoid-cell lineages might further

contribute to disease, through the production of pro-

inflammatory cytokines, such as IFN-γ. Counter-

regulatory T

2-cell responses might downregulate

disease and promote parasite clearance through anti-

body formation. Much severe pathology in malaria

therefore has an immunological basis, although this

remains poorly investigated for certain key processes

such as metabolic acidosis, which is the strongest

prognostic indicator of malarial fatality

and one

of the least understood aspects of pathogenesis. The

importance of immune processes in malaria patho-

genesis in humans is further exemplified by clear

associations of genetic polymorphisms in immune

loci — such as those encoding MBL, CD36, CD40

ligand, TNF, IFN-γ, IL-4 and the p40 subunit of IL-12

— with altered risk of disease

132

. Credible model sys-

tems allowing hypothesis testing by experimentation

show that multiple convergent factors are required,

and few components are sufficient, for disease pro-

gression, indicating that there are multiple potential

points of intervention in humans. Because T cells

contribute to these processes, vaccination strategies

should avoid exacerbating disease, by the induction of

appropriate counter-regulatory mechanisms or by the

neutralization of pathogenic parasite products.

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Acknowledgements

We thank A. Craig, V. Combes, N. Coltel, D. Hansen and

K. Evans for useful discussion. This work was supported by the

National Health and Medical Research Council (Australia),

the National Institutes of Health (United States), the Human

Frontier Science Program (France), the United Nations

Children’s Fund–United Nations Development Programme–

World Bank–World Health Organization Special Programme for

Research and Training in Tropical Diseases (Switzerland), the

Paludisme+ programme of the French Ministry of Research

(France), the Direction Générale des Armées (France), the

Fondation Recherche Médicale (France) and the Wellcome

Trust (United Kingdom). L.S. is an international research scholar

of the Howard Hughes Medical Institute (United States).

Competing interests statement

The authors declare no competing financial interests.

Online links

DATABASES

The following terms in this article are linked online to:

Entrez Gene:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

CD36 | EMP1 | ICAM1 | ICAM2 | IFN-γ | IL-1 | LFA1 | MAC1 |

P-selectin | P-selectin glycoprotein ligand 1 | TNF

Infectious disease information:

http://www.cdc.gov/ncidod/diseases/index.htm

malaria

FURTHER INFORMATION

The Walter and Eliza Hall Institute of Medical Research:

http://www.wehi.edu.au

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IMMUNOLOGY VOLUME 5

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Erratum: Low plasma concentrations of interleukin 10 in severe malarial anaemia compared with cerebral and uncomplicated malaria (The Lancet (1998) June 13 (1768))

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Jørgen Kurtzhals

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Jan 1999

Pathophysiology

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N.J. White

A summary of our present knowledge of the mechanism of immunity in malaria

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J.A. Sinton

The mechanism of immunity against malaria in communities living under hyperendemic conditions

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INDIAN J MED RES

S.R. Christophers

Platelet-derived interleukin 1 induces human endothelial adhesion molecule expression and cytokine production

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Oct 1991

C. M. Hawrylowicz

Interleukin 1 (IL-1) plays a central role in the regulation of the body's response to infectious and inflammatory stimuli. Recent evidence has shown that human platelets express a cell associated form of this proinflammatory cytokine very rapidly following activation. Since one of the earliest events in inflammation is frequently the rapid adhesion of platelets to injured endothelium, it was of interest to determine whether platelets express IL-1 in a functionally relevant form that can alter the phenotype of human endothelial cells in vitro. Thrombin activated platelets induced significant expression of the adhesion molecule intercellular adhesion molecule 1, as well as secretion of the IL-1 inducible cytokines IL-6 and granulocyte macrophage colony stimulating factor by cultured human umbilical cord and saphenous vein endothelial cells. This was inhibited by prior treatment of the platelets with antibody specific for IL-1. These results suggest that platelet delivered IL-1 might initiate and regulate some of the earliest phases of the inflammatory response. An additional observation of interest was differential induction of endothelial leucocyte adhesion molecule 1 by activated platelets on saphenous vein but not umbilical vein but not umbilical vein endothelial cells, which suggests functional heterogeneity of the endothelial cells.

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Article

May 2003
INT IMMUNOL

S. Hanum P.

A comparative study was carried out on cytokine and chemokine responses in a cerebral malaria (CM)-susceptible or -resistant strain of mice (C57BL/6 or BALB/c respectively) in Plasmodium berghei ANKA infection. C57BL/6 mice died by 10 days after infection when parasitemia was ∼15-20% with cerebral symptoms, while BALB/c mice survived until week 3 after infection. Although both strains showed T h 1-skewed responses on day 4 after infection, significantly higher levels of IFN-γ, tumor necrosis factor (TNF)-α and NO were observed during the course of the infection in BALB/c, suggesting that T h 1 responses are involved in the resistance. interestingly, in the brain, both strains expressed IFN-inducible protein of 10 kDa (IP-10) and monocyte chemotactic protein (MCP)-1 genes as early as at 24 h post-infection, whereas some differences were observed between both strains thereafter, i.e. enhanced expression of RANTES in C57BL/6, and of IFN-γ and TNF-a in BALB/c respectively. Moreover, the expression of IP-10 and MCP-1 genes in KT-5, an astrocyte cell line, was induced In vitro upon stimulation with a crude antigen of malaria parasites. These results suggest that the direct involvement of brain parenchymal cells takes place in response to plasmodial infection, providing a new aspect to analyze possible mechanisms of CM. This is the first report on the chemokine expression in neuroglial cells in response to malaria infection.

Immunological processes in malaria pathogenesis

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