| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
from Human NK Cells by Live Plasmodium falciparum-Infected Erythrocytes1
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production in response to human PBMC activation by
intact, infected RBC (iRBC) or freeze-thaw lysates of P.
falciparum schizonts. Infected erythrocytes induce a more rapid
and intense IFN- response from malaria-naive PBMC than do P.
falciparum schizont lysates correlating with rapid iRBC
activation of the CD3-CD56+ NK cell population
to produce IFN-. IFN-+ NK cells are detectable within
6 h of coculture with iRBC, their numbers peaking at 24 h in
most donors. There is marked heterogeneity between donors in magnitude
of the NK-IFN- response that does not correlate with mitogen- or
cytokine-induced NK activation or prior malaria exposure. The NK
cell-mediated IFN- response is highly IL-12 dependent and appears to
be partially IL-18 dependent. Exogenous rIL-12 or rIL-18 did not
augment NK cell IFN- responses, indicating that production of IL-12
and IL-18 is not the limiting factor explaining differences in NK cell
reactivity between donors or between live and dead parasites. These
data indicate that NK cells may represent an important early source of
IFN-, a cytokine that has been implicated in induction of various
antiparasitic effector mechanisms. The heterogeneity of this early
IFN- response between donors suggests a variation in their ability
to mount a rapid proinflammatory cytokine response to malaria infection
that may, in turn, influence their innate susceptibility to malaria
infection, malaria-related morbidity, or death from
malaria. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and TNF-
, are essential mediators of protective
immunity to erythrocytic malaria (1, 2); these cytokines
can derive from either the innate or adaptive arm of the immune
response. Given that these cytokines play a role in both immunity and
pathology of malaria (1, 2), it is important to quantify
the relative contribution of these two components of the immune
response to the proinflammatory response.
Resistance to rodent malaria is absolutely dependent on signals
mediated by IFN- (1), and the difference between lethal
and nonlethal infections depends on the ability of the mouse to mount
an early IL-12, IFN-, or TNF- response (2, 3, 4, 5).
TNF- and IFN- act synergistically to optimize NO production
(6), which is involved in parasite killing
(7). Similarly, in humans, IFN- production is
correlated with resistance to reinfection with Plasmodium
falciparum (8, 9) and protection from clinical
attacks of malaria (10), plasma TNF- and nitrogen oxide
levels are associated with resolution of fever and parasite clearance
(11, 12), and plasma TNF- and IFN- mediate loss of
infectivity of circulating gametocytes (13). Many vaccine
developers now regard IFN- production to be the hallmark of effector
T cell function for malaria (14, 15).
A recent critical review of the literature (16) concluded
that control of the early peak of parasitemia in murine malaria
infections was dependent on innate rather than adaptive cellular immune
mechanisms, raising important questions about the role of innate
immunity in control of human malaria. Enhanced NK cell activity in
spleens of mice infected with irradiated Plasmodium berghei
sporozoites was demonstrated many years ago (17); more
recently, Plasmodium yoelii sporozoite infection has been
shown to induce a rapid inflammatory response in the liver
characterized by NK cell, macrophage, and T cell infiltration and
IFN- production (18). It has been proposed that
protective immunity to P. yoelii liver stages mediated by
parasite-specific CD8+ T cells is dependent on
the presence of IL-12 and NK cells (19), indicating an
important synergy between innate and adaptive immunity in this system.
There is less information regarding the role of innate immune
mechanisms in controlling blood stage malaria infections; however,
depletion of NK cells from Plasmodium chabaudi-infected mice
results in a more severe course of infection with higher parasitemia
and increased mortality (20).
Regarding the human immune response to malaria, we and others have
shown that PBMC from malaria-unexposed donors can produce IFN- in
response to stimulation by either live or dead schizont Ags
(21, 22, 23, 24). Live parasites induce proliferation of both
and 
TCR+ T cells, whereas dead
schizont extract activates only TCR+ T
cells (24). These cells have been widely assumed to be the
major source of IFN- (23, 25). Activation of
TCR+ T cells has been ascribed to
reactivation of a polyclonal population of memory cells primed by
exposure to cross-reactive Ags (21, 26, 27), whereas the
TCR+ T cell response is restricted to the
V9V2 subset (28, 29), and is induced by small,
phosphorylated nonprotein Ags similar to those described for
mycobacteria (30). These findings are consistent with
cytokine responses to malaria Ags in unexposed donors being derived
from cells of the adaptive immune system. However, Scragg et al.
(31) have recently reported very early induction of
TNF-, IL-12, and IFN- (within 10 h) by live, parasitized
erythrocytes, and Hensmann et al. (32) have shown that
live parasites induce TNF- and IFN- from
V9+ T cells and TNF- from
CD14+ monocytes, within 18 h. Cytokine
induction is dependent on the presence of both monocytes and
lymphocytes (31), indicating that this is not a classical
endotoxin-like response as had previously been thought
(33). Increased NK-like cytotoxicity has been reported
during mild malaria infection (34), but appears to be
depressed in children with severe disease (35). Coculture
of PBMC with soluble malaria Ags has been reported to increase their
cytotoxic activity against an NK-sensitive cell line (36),
and CD3- CD56+ NK cells
have been reported to lyse schizont-infected erythrocytes
(37).
The purpose of this study therefore was to investigate the kinetics and
cellular origins of IFN- induced by P. falciparum malaria
in naive and malaria-exposed human blood donors, to determine the
contribution of cells of the innate and adaptive immune response to the
early inflammatory response. Our observation that
CD3-CD56+ NK cells are
major contributors to the first wave of IFN- production, within
24 h of PBMC coculture with parasitized erythrocytes, led us to
investigate the activation requirements for human NK cells responding
to malaria parasites.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
P. falciparum parasites of the 3D7 strain were grown in A+ human erythrocytes in RPMI 1640 (Life Technologies, Paisley, U.K.) with 25 mM HEPES (Sigma, Poole, U.K.), 1 µg/L hypoxanthine (Life Technologies), 28 mM sodium bicarbonate, and 10% normal AB human serum (pH 7.3). Flasks were gassed with 3% O2/4% CO2/93% N2 and incubated at 37°C; parasite development was monitored by examination of Giemsa-stained thin blood smears. Cultures were routinely screened for mycoplasma contamination by PCR (BioWhittaker, Wokingham, U.K.) and shown to be mycoplasma free. Mature schizonts were harvested from cultures of 6–7% parasitemia by centrifugation through 60% Percoll (Sigma-Aldrich, St. Louis, MO) and resuspended in culture medium. Schizonts were either placed directly into culture (live parasite Ag, infected RBC (iRBC)3) or freeze-thawed twice in liquid nitrogen to produce P. falciparum schizont extract (PfSL), which was stored at -70°C until used. Freeze-thawed uninfected erythrocytes (uRBC) were used as controls.
PBMC cultures
Thirty adult blood donors were recruited for the study over the
course of several months. Fifteen donors were of European descent, four
originated from the Indian subcontinent, and eleven were African.
Eighteen of the donors had never been infected with malaria, while the
remainder had been infected with malaria on one or more occasions in
the past. Details of all donors are shown in Table I
. Each donor’s NK cells were assayed
between two and five times on different days to verify the
reproducibility of their IFN- production, and results were found to
be consistent. Venous blood was collected into sterile, heparinized
tubes, and PBMCs were separated by density centrifugation (Lymphoprep;
Nycomed, Uppsala, Sweden). PBMCs were resuspended at 1 x
106 cells/ml in RPMI 1640 with 2 mM
L-glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin
(all Life Technologies), and 10% human AB serum (Sigma-Aldrich). Cells
were aliquoted at 106/well into 24-well
flat-bottom tissue culture plates (Nunclon, Paisley, U.K.) and
incubated with the appropriate Ag at 37°C in an atmosphere of 5%
CO2 for 1, 2, 4, or 6 days. In initial
experiments, iRBC, PfSL, and uRBC were tested for their ability to
induce IFN- over a range of concentrations (1 x
105 to 1 x 107
schizonts or RBC per well); the optimal concentration of malaria Ags
for induction of IFN- from cells of naive donors was found to be
3 x 106 parasites/106
PBMC (which is equivalent to a parasitemia of 15,000 iRBC/µl blood),
and this was used for all further assays. Growth medium was used as a
negative control and induced similar levels of IFN- as uRBC. A
mixture of human rIL-12 (rhIL-12) (1 ng/ml), rhIL-18 (40 ng/ml)
(PeproTech, London, U.K.), and rhIL-2 (2 ng/ml) (Boehringer Mannheim,
Lewes, U.K.), or the mitogen PHA (1 µg/ml; Difco/BD Biosciences,
Oxford, U.K.) was used as positive controls for NK cell activation (PHA
was not necessarily expected to act directly on NK cells, but to
activate other cells in the PBMC culture, inducing cytokines that would
lead to NK activation). To determine the effect of exogenous cytokines
on the response to malaria parasites, rIL-12 or rIL-18 were added to
cultures at concentrations ranging from 0.1 to 10.0 ng/ml.
|
Brefeldin A (10 µg/ml; Sigma-Aldrich) was added to cell
cultures for the last 3 h of incubation. After the appropriate
incubation period, supernatants were collected from each culture and
stored at -70°C for cytokine analysis by ELISA. Cells were washed
twice with FACS buffer (1x PBS, 0.1% NaN3,
0.1% BSA), resuspended in FACS buffer at a concentration of 5 x
105 cells/100 µl, and stained for 40 min at
4°C with appropriate combinations of labeled Abs. Cells were washed
twice with FACS buffer, fixed in 2% paraformaldehyde in PBS for 15 min
at room temperature in the dark, and washed once with FACS buffer.
Cells were resuspended in 100 µl permeabilization buffer (1x PBS,
1% saponin, 0.1% sodium azide) with anti-cytokine Ab, incubated
in the dark at 4°C for 30 min, and washed with FACS buffer. Finally,
cells were suspended in 300 µl FACS buffer and analyzed in a FACScan
flow cytometer (BD Biosciences). Data analysis was performed with
CellQuest software (BD Biosciences). A total of 100,000 events was
collected from each sample. The following mAbs were used: CD3 Tricolor
(TRI), CD8 TRI, IgG1 TRI (all Caltag Laboratories, Burlingame, CA);
TCR FITC, TCR FITC, CD56 FITC, CD4 FITC, IgG1 FITC, IgG2
FITC, V9 PE, IFN- PE, TNF- PE, and IgG1 PE (all BD
Biosciences); V2 FITC and V9 FITC (Serotec, Oxford, U.K.).
Cytokine ELISA
IFN- and IL-12 (p40 and p70) were detected in cell
supernatants by sandwich ELISA using commercially available reagents;
all samples were tested in duplicate according to the manufacturer’s
recommendations. IFN- Abs and standard were purchased from BD
Biosciences; IL-12 Abs and standard were from R&D Systems (Abingdon,
U.K.). Where samples gave values above the top of the standard curve,
supernatants were retested at 1/10 or 1/100 dilutions in RPMI 1640, and
cytokine levels were recalculated.
IL-12 and IL-18 neutralization
Neutralizing goat anti-human IL-12 polyclonal Ab (R&D Systems) was added to PBMC cultures at concentrations ranging from 0.5 to 10 µg/ml. An isotype-matched control Ab (goat IgG; Sigma-Aldrich) was used at the same concentrations. Neutralizing mouse polyclonal anti-human IL-18 or a mouse IgG1 control Ab (both R&D Systems) were used at concentrations from 0.1 to 5.0 µg/ml.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
response to P. falciparum by cells from
malaria-naive donors
We examined the kinetics of IFN- secretion from PBMC of five
European, malaria-naive donors in response to iRBC or PfSL, measuring
IFN- concentration in culture supernatants by ELISA (Fig. 1). uRBC induced minimal IFN-
production over a period of 6 days. Lysed parasites induced modest, but
statistically significant IFN- responses in cells from all donors.
Cells from three donors produced IFN- from about day 4; cells from
one donor made only a modest and transient response; and cells from one
other donor made high levels of IFN- within 24 h. In contrast,
live parasites rapidly induced high levels of IFN- production from
cells from all donors, with IFN- concentrations in excess of 10,000
pg/ml by day 4 and concentrations of up to 200,000 pg/ml by day 6.
Differences between PfSL-induced and iRBC-induced IFN-
concentrations were statistically significant at all time points
(Wilcoxon signed rank test, Z = -2.023, n =
5, p = 0.043). Thus, the IFN- response to iRBC was
more rapid, and maximal concentrations were 10-fold higher than the
response to PfSL.
|
production
We hypothesized that the rapid IFN- response to iRBC
represented triggering of a population of innately activated cells such
as NK cells, NK-T, or T cells. To determine the source of
IFN- production during the first 3 days of exposure of naive PBMC to
malaria Ags, we incubated PBMC with iRBC, PfSL, or uRBC and harvested
cells after periods of between 3 and 72 h for analysis of cell
surface phenotype and intracellular IFN- by flow cytometry.
IFN-+ cells were gated and analyzed for
expression of CD3, TCR, TCR, and CD56. As an example, data
for one donor after 24-h incubation of PBMC with 3 x
106 iRBC or uRBC or 3 x
106 lysed infected erythrocytes are shown in Fig. 2. For cells incubated with iRBC, IFN-
was derived from a mixed population of cells, of which the most
numerous are CD56+CD3- NK
cells. After subtraction of the background counts for the isotype
control Ab and uRBC control, 62% of the iRBC-induced
IFN-+ cells were
CD3-CD56+ NK cells; the
remainder were CD3+ T cells. After 24-h
incubation with PfSL, the number of cells staining for IFN- was
almost 4-fold lower than for iRBC; after subtraction of background
staining, 42% of the IFN-+ cells were found
to be NK cells.
|
, a and b).
Optimal NK cell IFN- production was observed at either 1 x
106 or 1 x 107
iRBC/106 PBMC. For additional experiments, intact
and lysed parasites were used at a final concentration of 3 x
106 parasites/106 PBMC in a
volume of 1 ml; control cultures contained 3 x
106 uRBC.
|
was examined over a period of
72 h (data from two representative donors are shown in Fig. 3
, c and d). iRBC-induced IFN- was detected
within NK cells as early as 6 h and peaked between 15 and 24
h in all donors. The kinetics of the iRBC-induced response was similar
to that of PHA-mediated NK activation. Twenty-four-hour cultures were
thus used to detect NK cell IFN- production in all future
assays.
As others (31, 32) have reported early production of
TNF- from PBMCs and as NK cells have been reported to make TNF-
(38), we also stained NK cells for intracellular TNF-.
As can be seen in Fig. 3, e and f, a small
percentage of NK cells could be induced to express TNF- after
incubation with PHA for 15–24 h, but few, if any, NK cells were
induced to secrete TNF- by incubation with iRBC (note the difference
in the scale on the y-axis for IFN- and TNF-).
To determine whether rapid induction of IFN- from NK cells by iRBC
was a universal phenomenon, we looked at the 24-h IFN- response in a
cohort of 30 human blood donors (Table I). The number of
IFN-+ cells/100,000 events was calculated for
PBMC cultured with uRBC, iRBC, or PfSL, and the number (%) of the
IFN-+ cells that were NK cells (i.e.,
CD3-, CD56+,
IFN-+) is also shown. In both the
malaria-unexposed and malaria-exposed donors, there is marked
heterogeneity in the number of IFN-+ cells in
both PfSL and iRBC cultures. In the malaria-unexposed donors, iRBC
induced significantly higher numbers of PBMC to produce IFN- than
did PfSL (Wilcoxon signed rank test, Z = 2.308,
p = 0.021), but this difference was not significant for
the malaria-exposed donors (Z = 1.177, p =
0.239). Similarly, in unexposed donors, iRBC induced significantly more
NK cells (in terms of both absolute numbers and percentages) to produce
IFN- than did PfSL (Z = 2.591, p = 0.009) and,
again, this difference was not significant for the malaria-exposed
donors (Z = 1.255, p = 0.209). The
total number of IFN-+ cells, and particularly
the number of IFN-+ NK cells, was somewhat
lower in the malaria-exposed donors than in the malaria-unexposed
donors, but there was considerable heterogeneity within the
malaria-exposed group and these differences were not statistically
significant (Student’s t test, t 
1.88,
p 
0.07 for all comparisons). Perhaps the most
noticeable trend in the data was the marked variation between donors in
the numbers of IFN-+ NK cells that were
induced by iRBC (ranging from 2,940 cells/100,000 events to less
than 20).
Taken together, these data suggest that: 1) the ability to mount a
rapid IFN- response to malaria parasites is partially dependent upon
the ability of NK cells to respond to malaria parasites; 2) the
difference in the magnitude of the response to iRBC and PfSL is due in
large part to the ability of iRBC to activate NK cells; and 3)
individuals vary in their ability to make an NK cell response to
malaria parasites.
Heterogeneity of the human NK cell response to malaria parasites
Variation between individuals in their T cell response to specific
Ags is commonplace and relatively easily explained by differences in T
cell repertoire, MHC genotype, and prior exposure to Ag. Substantial
variation in the NK cell response to a given stimulus was less expected
and less easy to explain, as this represents an innate response in
which prior exposure to the pathogen is not expected to augment the
response. Indeed, NK cells from malaria-exposed donors were somewhat
less likely to make IFN- than cells from naive donors (Table I and
Fig. 4). One possibility is that the
heterogeneity represents inherent differences between donors in the
ease with which their NK cells are activated to produce IFN-.
However, we found no obvious correlation between the response to iRBC
and the response to other stimuli such as recombinant cytokines or (as
shown in Fig. 4) PHA.
|
As activation of NK cells is known to be at least partially IL-12 and IL-18 dependent in many systems (39, 40, 41), we hypothesized that the difference between donors in their ability to make an NK cell response to malaria might be due to differences in their ability to make IL-12 or IL-18 in response to iRBC activation.
To determine whether NK cell responses to malaria were indeed IL-12 or
IL-18 dependent, we incubated PBMC with iRBC or uRBC for 24 h in
the presence or absence of increasing concentrations of neutralizing Ab
to human IL-12 or IL-18 (Fig. 5). The
percentage of IFN-+ NK cells was markedly
reduced in the presence of 0.5 µg/ml anti-human IL-12, but not by
equivalent concentrations of an isotype-matched Ab (Fig. 5a); NK cell responses were not further reduced by
increasing doses of Ab. Anti-IL-12 at a concentration of 0.5 µg/ml
consistently reduced the percentage of IFN-+
NK cells by between 50 and 100% (Fig. 5b). In a comparable
experiment, anti-IL-18 Ab had rather variable effects on the
proportion of NK cells that made IFN- in response to iRBC. In
titration experiments, anti-IL-18 Ab inhibited NK activation by up
to 50% at high concentrations (5.0 µg/ml) (Fig. 5c) and
was able to partially inhibit induction of IFN- in NK cells of some
donors (e.g., 094M and FMO), but not others. (e.g., 053M) (Fig. 5d).
|
in supernatants or the percentage of IFN-+ NK
cells after 24 h (data not shown). Also, addition of exogenous
rIL-12 did not enhance the response of low responding donors and did
not enhance PfSL-induced responses to the level of iRBC-induced
responses (data not shown). Thus, although IL-12 plays an essential
role in the activation of NK cells to make IFN-, lack of IL-12 is
insufficient to explain the differences in IFN- responses between
individuals or between live and dead parasites. In a comparable experiment, addition of rIL-18 to 24-h PBMC cultures with PfSL or iRBC had no effect on the NK cell response to PfSL, although low doses of rIL-18 (0.1 or 1.0 ng/ml) did marginally enhance the NK response to iRBC in most donors (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and TNF-) may enable the
infected host effectively to control the exponential replication of
blood stage parasites until the adaptive immune response can take over.
This might be of most benefit during a primary infection, but, given
the extent of antigenic polymorphism in malaria, innate responses may
also be required to control reinfections of variant genotype until
novel adaptive responses can be generated.
It has long been known that T cells from malaria-naive donors can
proliferate and secrete cytokines in response to P.
falciparum Ags. Responses tend to peak after 6–7 days activation
in vitro and the proliferating cells have been identified as either
TCR+ T cells, which respond in a classical
MHC-restricted manner to both live and dead parasite Ags, or
TCR+ T cells that respond preferentially to
live parasites (23, 24); both cell types have also been
shown to secrete IFN- (21, 23, 25). Recently, it has
been suggested that live P. falciparum can induce rapid
(within 12–24 h) IFN- and TNF- responses (31) and
that TCR+ T cells may contribute to the
early IFN- response (32). We have also recently
demonstrated an important role for IL-12 in malaria-induced IFN-
production (42). We have therefore conducted a detailed
study of the kinetics of IFN- production by P.
falciparum-activated PBMC.
The first interesting observation was that live parasites induce a much
stronger and much more rapid IFN- response than do dead parasites.
Differences between live and dead parasites have been noticed
previously, particularly in their ability to activate
TCR+ T cells (24, 32). We have
now extended this observation to include preferential induction of
IFN- production in NK cells by live parasitized erythrocytes. The
requirement for live parasites within intact RBC for induction of the
innate response may be due to a need for direct contact between the
parasitized red cell and the leukocyte (be it a lymphocyte or an
accessory cell), or may be due to instability of the parasite-derived
ligands that interact with cell surface receptors.
The nature of the malarial ligands that might be involved in induction
of the innate response is largely unknown. Scragg et al.
(31) have shown that early cytokine induction by P.
falciparum is not due to the presence of classical endotoxins:
levels of IL-12 and IL-10 induced by malaria Ags are orders of
magnitude lower than the levels induced by binding of bacterial LPS to
CD14, and a CD14+ monocyte-like cell line that
responds to a wide variety of bacterial endotoxins by secretion of
TNF- fails to respond to P. falciparum (31).
Malarial GPIs have been shown to activate macrophages and vascular
endothelium (33, 43). GPIs from Trypanosoma
cruzi have been shown to induce IL-12 via binding to Toll-like
receptor 2 (44), but nothing is currently known of
malarial ligands for Toll-like receptors. P.
falciparum-derived, phosphorylated, nonprotein moieties similar to
those isolated from mycobacteria have been shown to be ligands for
TCR+ T cells (30) and, due to
their highly lipophilic nature, are likely to have a rather short
t1/2 in solution. Finally, malarial
GPIs have also been reported to activate murine CD1d-restricted NK-T
cells (45), although these cells then produce IL-4 rather
than IFN-, but at least one study has failed to reproduce the
findings (46).
In this study, we have shown that CD3-
CD56+ NK cells are major contributors to the
early IFN- response to malaria parasites.
IFN-+ NK cells can be identified as early as
6 h after stimulation in some donors, and the peak of the NK
IFN- response occurs between 15 and 24 h. Preliminary studies
in our laboratory indicate that the activated NK cells undergo
apoptosis from 
24 h (K. Artavanis-Tsakonas and E. Riley, unpublished
observation). In the majority of donors, NK cells are the first
population to become IFN-+, with T
cells becoming IFN-+ after 48–72 h and
T cells beginning to make IFN- after 4–6 days (data not shown).
We were not surprised to find that the NK cell response was highly
IL-12 dependent; this is widely reported for NK cells
(39), and has been shown for NK responses to a number of
pathogens (40, 41). We considered the possibility that
differences between donors, and between Ag preparations, in the
magnitude of the IFN- response might be due to differences in IL-12
induction. However, addition of rIL-12 did not significantly enhance
the response of individual donors and did not increase the response to
PfSL to levels seen for iRBC.
The requirement for IL-18 was less clear, with some donors showing a
reduction in NK cell responses to iRBC in the presence of
anti-IL-18 Ab and cells from other donors being unaffected. In
contrast, low doses of rIL-18 did marginally enhance the proportion of
NK cells that could be induced to produce IFN- in response to iRBC
in most donors, indicating that IL-18 can indeed augment the NK
response to malaria parasites. Taking the IL-12 and IL-18 data
together, it appears that while monokines, produced for example by
dendritic cells or macrophages in the PBMC cultures, are required to
optimize the NK cell response to P. falciparum, these
monokines are not sufficient on their own to induce the levels of
IFN- production obtained with malaria-infected RBC. A lack of IL-12
or IL-18 in the culture medium is insufficient to explain the very low
levels of NK cell activation induced by parasite lysates or the very
low responses to iRBC in some donors. However, the influence of other
cytokines (e.g., IL-2 and IL-15) and the ability of an individual’s
macrophages or dendritic cells to respond to malaria Ags in other ways
require further investigation.
In addition to NK activation by cytokines, our data raise the
possibility that ligands expressed on the intact iRBC are specifically
recognized, either by the NK cell itself or by an APC population.
Preliminary data from our laboratory indicate that iRBC can bind to NK
cells and that contact between the NK cell and the iRBC is required for
optimal IFN- induction (K. Artavanis-Tsakonas and E. Riley,
unpublished data). Also, there is a report in the literature that human
NK cells can lyse parasite-infected RBC (37), indicating
that direct cell:cell contact does occur. Thus, optimal activation of
NK cells may require integration of two or more signals transduced
through different receptors.
It is clear that there is considerable variation between individuals in
the magnitude of their NK cell IFN- response. This heterogeneity
does not appear to be due to inherent differences in the ease of NK
activation or differences in IL-12 or IL-18 production. The difference
between malaria-exposed and malaria-unexposed donors is intriguing.
Although the numbers are quite small, and the difference not quite
statistically significant (t = 1.878,
p = 0.07), the numbers of NK cells making IFN- were
lower in iRBC-stimulated cultures from the exposed than the unexposed
donors. At this stage, we cannot say whether this represents
down-regulation of NK cell responses by acquired immune responses or is
due to genetic differences between the groups (10 of the 12 exposed
donors were Africans compared with only 1 of 18 in the unexposed
group). A possibility is that genetic variation in NK cell receptor
expression, particularly of activating killer cell Ig-like receptors
(47), might influence the response to malaria.
Importantly, our data suggest that the NK cells are a significant
source of IFN- in the first few hours of a malaria infection, and
thus may be an important component of the innate defense against
malarial parasitemia. Surprisingly, this innate response is not
universal among human blood donors, raising interesting questions
about the functional importance of the innate response to malaria
infection. Two opposing hypotheses can be proposed; rapid IFN-
production may be associated with efficient induction of
IFN--mediated effector mechanisms and enhanced ability to control
malaria infections. Alternatively, rapid innate production of IFN-
may predispose to overproduction of inflammatory cytokines and
increased risk of severe malaria. Studies in malaria endemic areas will
be required to determine which, if either, of these scenarios is
correct.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Eleanor M. Riley, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. E-mail address: eleanor.riley{at}lshtm.ac.uk
3 Abbreviations used in this paper: iRBC, infected RBC; PfSL, P. falciparum schizont lysate; rh, recombinant human; TRI, Tricolor; uRBC, uninfected RBC.
Received for publication May 20, 2002. Accepted for publication July 9, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor deficient mice. Parasite Immunol. 19:375.[Medline]
and TNF and occurs via a nitric oxide-dependent mechanism. J. Immunol. 155:2545.[Abstract]
in lethal and nonlethal malaria in susceptible and resistant hosts. J. Immunol. 143:2038.[Abstract]
interferon responses in lethal and nonlethal murine blood stage malaria. Infect. Immun. 65:1593.[Abstract]
expression in the spleen correlates with resistance to blood-stage malaria in mice. Infect. Immun. 64:535.[Abstract]
and interferon, but not by interleukin-4, during blood stage malaria in mice. Infect. Immun. 64:44.[Abstract]
interferon in serum against exoerythrocytic stages of Plasmodium falciparum. J. Clin. Microbiol. 29:1757. responses are associated with resistance to reinfection with Plasmodium falciparum in young African children. J. Infect. Dis. 179:980.[Medline]
+ malaria specific responses from non-exposed donors. Int. Immunol. 4:985. T cells respond to membrane-associated components of malaria-infected erythrocytes by proliferation and production of IFN. Immunology 88:412.[Medline]
+ T cells preferentially respond to live rather than killed malaria parasites. Infect. Immun. 66:2393.9+ T cells stimulated by Plasmodium falciparum. Parasite Immunol. 17:413.[Medline]
chain V segment usage. Int. Immunol. 8:1877.9 subset of human T cells. Eur. J. Immunol. 21:2613.[Medline]
9 V2 T cells following stimulation of peripheral blood lymphocytes with extracts of Plasmodium falciparum. Int. Immunol. 4:361. T cells are related to the phosphorylated antigens of mycobacteria. Infect. Immun. 64:2892.[Abstract]
production and antiviral defense. J. Immunol. 156:1138.[Abstract]
in response to bacterial pathogens. J. Immunol. 166:1097.This article has been cited by other articles:
|
|
 |
|
  L. J. Robinson, M. C. D'Ombrain, D. I. Stanisic, J. Taraika, N. Bernard, J. S. Richards, J. G. Beeson, L. Tavul, P. Michon, I. Mueller, et al. Cellular Tumor Necrosis Factor, Gamma Interferon, and Interleukin-6 Responses as Correlates of Immunity and Risk of Clinical Plasmodium falciparum Malaria in Children from Papua New Guinea Infect. Immun., July 1, 2009; 77(7): 3033 - 3043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. S. Franklin, P. Parroche, M. A. Ataide, F. Lauw, C. Ropert, R. B. de Oliveira, D. Pereira, M. S. Tada, P. Nogueira, L. H. P. da Silva, et al. Malaria primes the innate immune response due to interferon-{gamma} induced enhancement of toll-like receptor expression and function PNAS, April 7, 2009; 106(14): 5789 - 5794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Ing and M. M. Stevenson Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice Infect. Immun., February 1, 2009; 77(2): 770 - 782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Kim, S. Parikh, J. C. Sun, A. Myrick, L. L. Lanier, P. J. Rosenthal, and J. L. DeRisi Experimental Malaria Infection Triggers Early Expansion of Natural Killer Cells Infect. Immun., December 1, 2008; 76(12): 5873 - 5882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Siddiqui, H. Bora, N. Singh, A. P. Dash, and Y. D. Sharma Expression, Purification, and Characterization of the Immunological Response to a 40-Kilodalton Plasmodium vivax Tryptophan-Rich Antigen Infect. Immun., June 1, 2008; 76(6): 2576 - 2586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Othoro, J. M. Moore, K. A. Wannemuehler, S. Moses, A. Lal, J. Otieno, B. Nahlen, L. Slutsker, and Y. P. Shi Elevated Gamma Interferon-Producing NK Cells, CD45RO Memory-Like T Cells, and CD4 T Cells Are Associated with Protection against Malaria Infection in Pregnancy Infect. Immun., April 1, 2008; 76(4): 1678 - 1685. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Roy, P. F. Barnes, A. Garg, S. Wu, D. Cosman, and R. Vankayalapati NK Cells Lyse T Regulatory Cells That Expand in Response to an Intracellular Pathogen J. Immunol., February 1, 2008; 180(3): 1729 - 1736. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. B. McCall, M. G. Netea, C. C. Hermsen, T. Jansen, L. Jacobs, D. Golenbock, A. J. A. M. van der Ven, and R. W. Sauerwein Plasmodium falciparum Infection Causes Proinflammatory Priming of Human TLR Responses J. Immunol., July 1, 2007; 179(1): 162 - 171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Hansen, N. J. Bernard, C. Q. Nie, and L. Schofield NK Cells Stimulate Recruitment of CXCR3+ T Cells to the Brain during Plasmodium berghei-Mediated Cerebral Malaria J. Immunol., May 1, 2007; 178(9): 5779 - 5788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Walther, J. Woodruff, F. Edele, D. Jeffries, J. E. Tongren, E. King, L. Andrews, P. Bejon, S. C. Gilbert, J. B. De Souza, et al. Innate Immune Responses to Human Malaria: Heterogeneous Cytokine Responses to Blood-Stage Plasmodium falciparum Correlate with Parasitological and Clinical Outcomes J. Immunol., October 15, 2006; 177(8): 5736 - 5745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Roland, V. Soulard, C. Sellier, A.-M. Drapier, J. P. Di Santo, P.-A. Cazenave, and S. Pied NK Cell Responses to Plasmodium Infection and Control of Intrahepatic Parasite Development J. Immunol., July 15, 2006; 177(2): 1229 - 1239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Tripathi, D. J. Sullivan, and M. F. Stins Plasmodium falciparum-Infected Erythrocytes Increase Intercellular Adhesion Molecule 1 Expression on Brain Endothelium through NF-{kappa}B. Infect. Immun., June 1, 2006; 74(6): 3262 - 3270. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Mavoungou Interactions between natural killer cells, cortisol and prolactin in malaria during pregnancy. Clin. Med. Res., March 1, 2006; 4(1): 33 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Korbel, K. C. Newman, C. R. Almeida, D. M. Davis, and E. M. Riley Heterogeneous Human NK Cell Responses to Plasmodium falciparum-Infected Erythrocytes J. Immunol., December 1, 2005; 175(11): 7466 - 7473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Baratin, S. Roetynck, C. Lepolard, C. Falk, S. Sawadogo, S. Uematsu, S. Akira, B. Ryffel, J.-G. Tiraby, L. Alexopoulou, et al. Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum PNAS, October 11, 2005; 102(41): 14747 - 14752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Mitchell, A. M. Hansen, L. Hee, H. J. Ball, S. M. Potter, J. C. Walker, and N. H. Hunt Early Cytokine Production Is Associated with Protection from Murine Cerebral Malaria Infect. Immun., September 1, 2005; 73(9): 5645 - 5653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Hansen, K. J. Evans, M. C. D'Ombrain, N. J. Bernard, A. C. Sexton, L. Buckingham, A. A. Scalzo, and L. Schofield The Natural Killer Complex Regulates Severe Malarial Pathogenesis and Influences Acquired Immune Responses to Plasmodium berghei ANKA Infect. Immun., April 1, 2005; 73(4): 2288 - 2297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mavropoulos, G. Sully, A. P. Cope, and A. R. Clark Stabilization of IFN-{gamma} mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells Blood, January 1, 2005; 105(1): 282 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C.-F. Hsieh, A. Loukas, A. M. Wahl, M. Bhatia, Y. Wang, A. L. Williamson, K. W. Kehn, H. Maruyama, P. J. Hotez, D. Leitenberg, et al. A Secreted Protein from the Human Hookworm Necator americanus Binds Selectively to NK Cells and Induces IFN-{gamma} Production J. Immunol., August 15, 2004; 173(4): 2699 - 2704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Struik, F. M. Omer, K. Artavanis-Tsakonas, and E. M. Riley Uninfected erythrocytes inhibit Plasmodium falciparum-induced cellular immune responses in whole-blood assays Blood, April 15, 2004; 103(8): 3084 - 3092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hatabu, S.-i. Kawazu, M. Aikawa, and S. Kano Binding of Plasmodium falciparum-infected erythrocytes to the membrane-bound form of Fractalkine/CX3CL1 PNAS, December 23, 2003; 100(26): 15942 - 15946. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. M. Omer, J. B. de Souza, P. H. Corran, A. A. Sultan, and E. M. Riley Activation of Transforming Growth Factor {beta} by Malaria Parasite-derived Metalloproteinases and a Thrombospondin-like Molecule J. Exp. Med., December 15, 2003; 198(12): 1817 - 1827. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Artavanis-Tsakonas, K. Eleme, K. L. McQueen, N. W. Cheng, P. Parham, D. M. Davis, and E. M. Riley Activation of a Subset of Human NK Cells upon Contact with Plasmodium falciparum-Infected Erythrocytes J. Immunol., November 15, 2003; 171(10): 5396 - 5405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Su, M.-F. Tam, D. Jankovic, and M. M. Stevenson Vaccination with Novel Immunostimulatory Adjuvants against Blood-Stage Malaria in Mice Infect. Immun., September 1, 2003; 71(9): 5178 - 5187. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
who have varying levels of prior malaria exposure.
*, No IFN-