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Secretion and Restriction of Leishmania Growth1
* National Center for Cell Science, Ganeshkhind, India; and
Department of Life Science and Biotechnology, Jadavpur University, Calcutta, India
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Ab inhibit the anti-leishmanial
function of neutrophils, suggesting that the neutrophil-macrophage
interaction via CD28-CD80/CD86 plays an important role in the
IFN--dependent restriction of the parasite growth. Cross-linking of
neutrophil-expressed CD28 by monoclonal anti-CD28 Ab or B7.1-Ig or
B7.2-Ig results in phosphatidylinositol 3-kinase association with CD28
and in wortmannin-sensitive but cyclosporin A-resistant induction and
secretion of IFN-. Whereas the neutrophils secrete IFN- with CD28
signal alone, the T cells do not secrete the cytokine in detectable
amounts with the same signal. Thus, neutrophil-expressed CD28 modulates
not only the granulocyte migration but also induction and secretion of
IFN- at the site of infection where it migrates from the
circulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was reported earlier that neutrophils clear Leishmania infection by virtue of their phagocytic function (6). Because Leishmania is a protozoan parasite that resides and replicates within the macrophages, the parasite clearance by phagocytosis alone as described in previous studies limits the role of neutrophils in Leishmania infection. It was proposed recently that neutrophils may interact with macrophages through cell surface molecules like LFA-1 during infection and inflammatory reactions (7). The macrophage surface molecules, CD80 and CD86, the receptors for CD28/CD152 (CTLA-4) on T cells, were shown to play important roles in T cell response against both Leishmania major and L. donovani infections (8, 9). Because we have already demonstrated CD28 expression on human peripheral blood neutrophils and CD28-regulated neutrophil migration in response to IL-8 (10), we investigated whether neutrophil-expressed CD28 interacts with CD80/CD86 on macrophages, resulting in alteration of cellular functions.
In this work we demonstrate that L. major infection of
macrophages results in increased IL-8 secretion and IL-8-directed
migration of neutrophil toward the L. major-infected
macrophages. The neutrophils interact with the macrophages through the
CD28-CD80/CD86 pathway, resulting in phosphatidylinositol 3-kinase
(PI3-kinase)3-dependent
induction and secretion of IFN-, which in turn controls L.
major growth in the macrophages. In addition, IFN- induction in
neutrophils does not require a primary signal as in T cells or NK
cells. Therefore, taken together, our results suggest that the
neutrophil-expressed CD28 serves an anti-leishmanial function by
interacting with the Leishmania-infected macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following approval from the Institutional Ethics Committee, venous blood from healthy volunteers was collected in heparin and cells were isolated from the blood using Polymorphoprep (Nycomed, Oslo, Norway) as described previously (10). PBMCs were plated at a final concentration of 2 x 106 cells/ml in RPMI 1640 supplemented with 10% FCS. Nonadherent lymphocytes were washed after 12 h. Monocytes were allowed to differentiate to macrophages for 72 h with washing every 24 h to remove the nonadherent cells. The cells recovered after the end of culture were >99% macrophages as judged by morphology, histochemistry, and FACS analysis (data not shown). Characterization of the neutrophils by morphology and histochemistry showed that >99.5% of the isolated cells were polymorphonuclear and that no CD3+ T cells were detectable (10). Cells were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) with 10% FCS.
CD4+ T cells were isolated from the PBMC fraction by affinity chromatography using the IsoCell human T cell isolation kit (Pierce, Rockford, IL), and the purity (>99.5%) was confirmed by FACS analysis of the anti-CD3-FITC-stained cells in FACSVantage (BD Biosciences, Mountain View, CA).
Quantitation of IL-8 in L. major-infected macrophage culture supernatant
Macrophages were infected with different doses of L. major promastigote (strain MHOM/SU/73/5ASKH). Extracellular parasites were washed after 4 h. The culture supernatant collected at different intervals after infection was assayed for IL-8 content by ELISA using a human IL-8 ELISA kit (Amersham Life Sciences, Little Chalfont, U.K.) following the manufacturer’s instructions.
Leishmanicidal assay
Human monocyte-derived macrophages were infected with L.
major at a 1:5 ratio for 4 h and were cocultured with
neutrophils at a 1:4 ratio in the presence of neutralizing
anti-IFN- Ab, CTLA-4-Ig (R&D Systems, Minneapolis, MN), control
human Ig, or other treatments, as indicated, for 36 h. The
intracellular parasite load was determined after staining the cells
with Giemsa stain. Five hundred cells per culture were counted. The
percentage of infection and number of parasites per 100 infected
macrophages were calculated.
Neutrophil transmigration assay
Neutrophil transmigration assay was performed using 24-well transwell inserts of 3-µm pore size (Falcon; BD Labware, Franklin Lakes, NJ), as described earlier (10). In brief, uninfected and L. major-infected macrophages were cultured in 24-well plates for 12 h. In some experiments, neutralizing anti-IL-8 Ab or control isotype (BD PharMingen, San Diego, CA) (10 µg/ml) were added to the lower well 30 min before placing the transwell insert. A total of 105 neutrophils, pretreated with anti-IL-8R A and anti-IL-8R B or control isotype (BD PharMingen) at 4°C for 30 min, were added to each insert and were allowed to migrate for 60 min. The filter was stained with Giemsa and cells in the lower surface were counted by randomly selecting 10 fields at x40 magnification (10).
Quantitation of IFN- in the culture supernatants by ELISA
CD28 on neutrophils was cross-linked using anti-CD28 Ab or
control isotype (BD PharMingen) as described earlier (10),
and the cells were cultured in the presence of indicated concentrations
of wortmannin (ICN Biomedicals, Costa Mesa, CA) or cyclosporin A
(Sigma-Aldrich, St. Louis, MO). The culture supernatant was assayed for
IFN- content by ELISA using the OptEIA kit (BD PharMingen) following
the manufacturer’s recommendations. CD4+ T cells
were cultured either in the presence of only anti-CD28 (5 µg/ml)
or along with immobilized anti-CD3 Ab (1 µg/ml). Supernatant was
collected at different time points and ELISA was performed as described
above. In parallel experiments neutrophils were also stimulated either
with CD28 alone or with anti-CD3 and anti-CD28 mAbs. To compare
the effect of IL-12 on IFN- induction by neutrophils and T cells,
cells were stimulated with indicated concentrations of IL-12 and
anti-CD28 mAbs. Culture supernatant was collected at different time
points and ELISA was performed using the OptEIA kit (BD PharMingen)
following the manufacturer’s recommendations.
Flow cytometry
Human monocyte-derived macrophages were infected with L. major at a 1:5 ratio. Six hours after infection, the cells were incubated with anti-human CD80-PE, anti-human CD86-FITC, and isotype-matched Abs (BD PharMingen) for 30 min in staining buffer (PBS containing 2% FCS and 0.1% sodium azide) at 4°C. This was followed by two washes in the staining buffer. The samples were analyzed using a FACSVantage flow cytometer.
For intracellular staining, the wells in a 96-well tissue culture plate
were coated with anti-CD28 mAb (10). Neutrophils were
plated to cross-link with the plate-bound anti-CD28 Ab
(10). Neutrophils were recovered from the wells, fixed and
permeabilized using Cytofix/Cytoperm Plus kit (BD PharMingen), and
stained for IFN- with FITC-labeled anti-IFN- Ab (BD
PharMingen). An isotype-matched FITC-conjugated Ab was used as isotype
control. The samples were analyzed using a FACSVantage flow
cytometer.
RT-PCR
Using TRIzol (Life Technologies), total RNA was isolated from
neutrophils after 4 h of incubation with medium alone or
stimulation with anti-CD28 mAb or isotype-matched Ab in the
presence or absence of wortmannin (10 ng/ml). For cDNA synthesis, 1
µg of total RNA from each sample was incubated with random primer,
0.1 M DTT, 500 µM dNTPs, 40 U RNase inhibitor, and 1 µl (400 U) of
Moloney murine leukemia virus reverse transcriptase (Life
Technologies). Samples were then incubated at 37°C for 1 h
followed by a 5-min incubation at 95°C. Amplification of synthesized
cDNA from each sample was conducted with DNAzyme DNA polymerase
(Finnzymer, Espoo, Finland) in 50 µl under following conditions:
95°C for 2 min, 94°C 1 min, 55°C for 1 min, and 72°C for 1 min
for a total of 35 cycles. Specific primers were designed to amplify the
human IFN- coding region (sense, 5'-GCAGGTCATTCAGATGTAGC-3';
antisense, 5'-GGCCCCTGAGATAAAGCC-3'). Each sample was also amplified
for either GAPDH or human 
-actin to ensure equal input of
cDNA.
CD28 immunoprecipitation and immunoblotting of PI3-kinase
Purified neutrophils were stimulated with immobilized anti-CD28 Ab (10 µg/ml) for 10 min at 37°C. The reaction was stopped by adding Triton X-100 lysis buffer (pH 7.5) containing 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin for 1 h at 4°C. The lysates were centrifuged at 10,000 x g at 4°C for 30 min. Supernatants were precleared with 5 µg of mouse IgG and 30 µl of protein G-Sepharose (Life Technologies) and were incubated with 5 µg monoclonal anti-CD28 Ab overnight at 4°C. Immune complexes were recovered by incubation with 30 µl protein G-Sepharose for 1 h at 4°C. The beads were washed five times with the lysis buffer and the immune complexes were resuspended in reducing Laemmli sample buffer (Sigma-Aldrich). The samples were heated to 95°C for 3 min and run on 10% SDS-PAGE. The separated proteins were transferred onto a nitrocellulose membrane. The membrane was incubated with blocking buffer containing 2% BSA and 0.1% Tween 20 in TBS (100 mM Tris-HCl (pH 7.5), 0.9% NaCl) for 1 h at room temperature, followed by incubation with rabbit anti-PI3-kinase polyclonal Ab at a 1/500 dilution in the same buffer containing 1% BSA and 0.1% Tween for 1 h. After washing, the blot was incubated for 1 h with HRP-conjugated anti-rabbit Ab at 1/2000 dilutions. Immunoblot signal was detected by using the ECL kit (Amersham Pharmacia Biotech, Little Chalfont, U.K.) following the manufacturer’s instructions.
Confocal microscopy
Confocal microscopy of neutrophils for checking IFN-
production was performed following the methods of Yeaman et al.
(11). Highly purified neutrophils were cross-linked with
anti-CD28 or isotype control Abs in the presence or absence of
wortmannin (10 ng/ml) for 5 h. The culture was continued for an
additional 4 h with brefeldin A. The cells were then stained with
1 µg of FITC-labeled anti-human IFN- Ab. After counterstaining
the nuclei with propidium iodide, cells were examined with a Zeiss LSM
510 confocal microscope equipped with argon and helium lasers
(Zeiss, Jena, Germany). Laser power, photomultiplier tube gains,
and confocal thresholds were set using long-pass and band-pass filters.
The physical parameters defined as above were kept constant throughout
the acquisition and analysis of the samples.
Statistical significance
The raw scores were analyzed using SigmaStat software program (Binary Semantics, Pune, India). The data are presented as mean ± SD of triplicates in one representative of at least three individual experiments. The fiducial limit for considering any difference of mean as significant is fixed at 0.01. The correlation coefficient for the association between parasite dose and IL-8 secretion was determined by Pearson’s r test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because Leishmania infection is known to induce
neutrophil infiltration to the site of infection (4), we
investigated whether L. major-infected macrophages secreted
IL-8, a major neutrophil-activating chemokine. Whereas infection of
macrophages with L. major promastigotes at ratios of 1:5,
1:10, and 1:20 resulted in the peak IL-8 secretion 
6 h after
infection, infection at a 1:1 ratio yielded the peak IL-8 concentration
24 h after infection (Fig. 1
A). With 1:1 and 1:5
macrophage:Leishmania infection ratios, the level of IL-8 in
the macrophage culture supernatant was undetectable 3 h after
infection. At higher macrophage:parasite ratios, the chemokine was
detectable after 3 h but was still significantly less than the
value observed 6 h after Leishmania infection (data not
shown). Because the peak IL-8 concentrations 6 h after infection
were directly proportional to the load of infection (correlation
coefficient = +0.954), the IL-8 secretion by L.
major-infected macrophages was parasite dose dependent. Thus, our
data are consistent with a previous observation describing the
appearance of IL-8 mRNA by RT-PCR 1 h after L. major
infection (12).
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B). The neutrophil migration was significantly inhibited by
both anti-IL-8 (Fig. 1B; p < 0.01) and
anti-IL-8R (Fig. 1C; p < 0.001) Abs,
suggesting that IL-8 secreted by the L. major-infected
macrophages chemoattract neutrophils toward the site of infection.
However, the incomplete inhibition of neutrophil migration in both
anti-IL-8 and anti-IL-8R blockade could be due to
chemoattraction effected by other chemokines released by the infected
macrophages and IL-8R recycling to the surface, as the migration assay
was performed at 37°C. Nevertheless, 65% inhibition of neutrophil
migration by IL-8 neutralization or by blocking IL-8Rs identified IL-8
as the major neutrophil chemoattractant released by the L.
major-infected macrophages. Neutrophils interact with macrophages via CD28-CD80/CD86
It is known that T cell-expressed CD28 interacts with CD80 or
CD86, also called B7.1 and B7.2, respectively, to generate the CD28
signal in T cells (13). Likewise, neutrophil-expressed
CD28 may also interact with CD80/CD86 on macrophages. To test that
hypothesis, we cocultured neutrophils with L. major-infected
macrophages in the presence or absence of CTLA4-Ig, a fusion protein
that blocks the CD28-CD80/CD86 interaction (14). The
culture containing CTLA4-Ig had higher number of parasites (Fig. 2, A and B) and
less IFN- (Fig. 2C), suggesting that the blockade of
CD28-CD80/CD86 interaction resulted in the inhibition of the
neutrophils’ anti-leishmanial function. The inhibition of the
leishmanicidal effect of neutrophils by anti-IFN- Ab suggested
that the CD28 signaling in neutrophils resulted in the secretion of
IFN-, which mediated the anti-leishmanial function of
neutrophils. In addition, the supernatant from CD28-cross-linked
neutrophil culture restricted the growth of L. major in
macrophages, and neutralization of IFN- in the supernatant reduced
the anti-leishmanial effect of the supernatant significantly (data
not shown).
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D). Thus, the profile of CD80 and CD86 expression early
after L. major infection validates our observation that the
neutrophils and macrophages interact through CD28-CD80/CD86 to express
the IFN--dependent anti-leishmanial function.
It has been argued that neutrophils, being active phagocytes, control
Leishmania infection by phagocytosis (6). In
this study, phagocytosis of Leishmania was not a significant
factor, because neutrophils were added to the culture after
extracellular parasites were washed off following the infection of
macrophages with Leishmania. We also investigated whether
CD28 cross-linking, which imparts anti-leishmanial function to
neutrophils by inducing cytokine secretion, increased phagocytosis.
Using Plasmodium-infected human RBC and
Leishmania as the models, we did not observe any difference
between phagocytosis by CD28 and isotype cross-linked neutrophils (data
not shown). This suggests that CD28-induced Leishmania
killing by neutrophils is not via parasite phagocytosis. Instead, the
fact that the parasite growth is inhibited by anti-IFN- Ab
demonstrates the IFN--dependent control of Leishmania
growth within macrophages.
Neutrophil-expressed CD28 interacts comparably with CD80 and CD86
to induce IFN-
Because Leishmania-infected macrophages showed an
increase in CD86 but not CD80 expression (Fig. 2D), and
because the regulation of T cell function was reported to be
differentially modulated by CD80 and CD86 (15, 16), we
examined whether neutrophil-expressed CD28 preferred CD86 to CD80 for
inducing IFN- production. CD28 on neutrophils was cross-linked with
immobilized B7.1-Ig-Fc and B7.2-Ig-Fc chimeras, the fusion proteins
created by fusion of the extracellular domains of either B7.1 or B7.2
to the Fc portion of human IgG1, and assayed for IFN- in the
supernatant after 12 h. Stimulation of neutrophils with human
B7.1-Ig-Fc chimera or B7.2-Ig-Fc chimera did not show a significant
difference in either IFN- secretion (Fig. 3A) or IFN- mRNA induction
(Fig. 3B). Similarly, although both the anti-CD80 and
anti-CD86 Abs alone prevented the IFN--mediated restriction of
parasite load in the neutrophil-macrophage coculture, they did not show
a marked difference in their ability to prevent anti-leishmanial
activity (data not shown). Our result thus indicates that neutrophils
do not show any preference for either of the two B7 molecules.
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secretion
Because the above experiments suggested that, upon CD28
cross-linking, neutrophils secrete IFN-, we investigated the
signaling involved in CD28-induced IFN- secretion. We found that
cross-linking with anti-CD28 Ab but not with the control isotype
resulted in IFN- secretion, which peaked within 6 h of
stimulation (Fig. 4A).
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secretion
from neutrophils, the cells were incubated with different doses of
wortmannin, followed by CD28 cross-linking. It was recorded that the
CD28-induced IFN- secretion was inhibited by wortmannin in a
dose-dependent manner (Fig. 4A, inset), suggesting a
PI3-kinase-dependent IFN- secretion. The observation was confirmed
by confocal microscopy, which showed that the CD28 cross-linked
neutrophils expressed more IFN- after 9 h than the isotype
cross-linked neutrophils and that wortmannin treatment of the CD28
cross-linked neutrophils inhibited IFN- production (Fig. 4B). We investigated whether CD28 was physically associated
with PI3-kinase after cross-linking, as described in case of T cells
(17). Immunoprecipitation of CD28 from the CD28
cross-linked neutrophils followed by Western blot with
PI3-kinase Ab showed the presence of the reported PI3-kinase protein
(18, 19, 20) only in CD28 cross-linked but not in the isotype
cross-linked or unstimulated neutrophils (Fig. 4C). The
result suggested that neutrophil-expressed CD28 associated with
PI3-kinase after CD28 cross-linking.
Although IFN- secretion was wortmannin sensitive, addition of
different concentrations of cyclosporin A to the culture did not
inhibit either transcription (Fig. 4D, inset) or secretion
(Fig. 4D) of IFN-, indicating the cyclosporin A
insensitivity of CD28-induced IFN- production in neutrophils as
reported in case of T cells (13). The observation thus
demonstrated that upon CD28 cross-linking PI3-kinase was associated
with CD28 to transmit the signal inducing IFN- secretion.
Neutrophils do not require a primary signal for CD28-induced
IFN- secretion
Because CD28 enhances the TCR-induced cytokine mRNA in T cells
(21), we checked whether CD28-induced IFN- secretion
was enhanced by a concomitant stimulation with IL-12, an
IFN--inducing cytokine. IL-12 addition to the
anti-CD28-stimulated neutrophil culture did not augment
CD28-induced IFN- secretion from neutrophils (Fig. 5A). Addition of anti-CD3
to neutrophils had no effect on anti-CD28-induced IFN- secretion
(Fig. 5B). In T cells, CD28-derived signal alone failed to
induce IFN- (Fig. 5C), although it enhanced
anti-CD3-induced IFN- production (Fig. 5D). A similar
kind of synergism between the CD28 signal and LPS or IL-8, the known
neutrophil activators, in IFN- induction was not observed in
neutrophils (data not shown). Besides differential signaling
requirements for IFN- production, the results described in this work
showed two other differences in the cytokine secretion by T cells and
neutrophils. Whereas the neutrophil IFN- secretion peaked 6–9 h
after stimulation, the T cell cytokine secretion peaked 48 h after
stimulation. Moreover, IFN- produced by T cells was 20 times more
than that produced by neutrophils.
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gene
transcription
Because IFN- secretion peaked rapidly after CD28 signaling, it
was possible that CD28 signaling through PI3-kinase induced IFN-
secretion from its preformed pool. Therefore, we stained intracellular
IFN- at various time points after CD28 cross-linking. We observed
that the intracellular IFN- staining was not detectable above the
background level until 3 h after CD28 signaling, suggesting that
the cytokine was induced and secreted but not released from the
preformed pool (Fig. 6A).
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gene transcription, the neutrophils were stimulated with anti-CD28
Ab in the presence of different doses of actinomycin D, an RNA
transcription inhibitor (22). We observed an actinomycin D
dose-dependent inhibition of IFN- production (Fig. 6B).
In other experiments, the optimum dose of actinomycin D (50 ng/ml) was
added to the anti-CD28-stimulated neutrophil cultures at different
time points after initiating the culture as indicated in Fig. 6C. Addition of the transcription inhibitor 3 h after
initiation of culture had little effect on the cytokine production
(Fig. 6C). These experiments suggested that transcription
was necessary for IFN- production after CD28 signaling.
To determine whether the PI3-kinase signaling was inducing
IFN- gene, RNA was prepared from the
neutrophils cross-linked with CD28 in the presence or absence of
wortmannin. Subsequent reverse transcription of the RNA, followed by
PCR, showed that the message for IFN- was extremely low in the
unstimulated neutrophils and that the message was increased
significantly after CD28 cross-linking but not isotype cross-linking
(Fig. 6D). Addition of wortmannin to the CD28 cross-linked
neutrophils reduced IFN- RNA levels to that found in unstimulated
neutrophils (Fig. 6D). These observations together indicate
that the IFN- gene is induced by CD28 signaling
through PI3-kinase and that the transcription is an obligatory
prerequisite for the cytokine production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4+V
8+ T cells
(23), which produces IL-4, to set the bias toward Th2
cells in the susceptible host (24). In contrast, an
earlier suggestion was that the initial T cell activation results in
the production of a wide array of cytokines (25) that
control parasite growth by regulating the macrophage activation or
inactivation (26) or by setting a Th subset bias
(24). Therefore, the time of involvement of T cells in the
initiation of the anti-leishmanial immune response is not well
defined, raising a possibility of involvement of non-T cells in the
initiation of anti-leishmanial immune response. Among the non-T
cells, neutrophils are known to infiltrate the site of infection as
early as 21 h after s.c. introduction of the parasite in mice
(our unpublished observation) and are present in the lesions of
cutaneous leishmaniasis patients (27). A possible role of
neutrophils at the site of lesion is phagocytosis of
Leishmania (6). Therefore, we tested the
hypothesis that neutrophils may play a crucial role in the initiation
of anti-leishmanial immune response.
In this paper, we describe how the inflammatory reaction against
Leishmania infection is initiated with the release of
chemokines such as IL-8 by Leishmania-infected macrophages,
followed by IL-8-dependent recruitment of neutrophils to the site of
infection. CD28 on neutrophils interacts with CD80/CD86 on macrophages
to generate CD28 signal-inducing IFN- secretion. In addition to
restriction of Leishmania growth, IFN- may alter the
cytokine microenvironment at the site of infection, affecting the IL-4-
and IFN--secreting T cell differentiation as a consequence (4, 28). Although neutrophils have already been reported to secrete
IFN- (11), our study provides the first mechanistic
description of how a signal generated by CD28 on cell surface regulates
the cytokine gene transcription and secretion in neutrophils. While
observations reported in this work indicate the significance of CD28
signaling in neutrophil-mediated induction of anti-leishmanial
immune response, earlier observations on murine L. major
infection in CD28-deficient mice suggested a limited role for CD28 in
Th subset differentiation during the infection (29, 30).
The apparent discrepancy between these observations can be explained by
a possible absence of CD28 on murine neutrophils, as casein-elicited
early peritoneal exudate cells do not stain for CD28 (data not shown).
Thus, although the current hypothesis for initiation of the disease
leishmaniasis by setting a Th subset bias relies heavily on T
cell-secreted cytokines (31), our observations define for
the first time a significant role of neutrophils in the afferent limb
of the anti-leishmanial immune response, especially that which
precedes the T cell response (32).
In the afferent arm of an immune response, interaction between the
cells of the adaptive immune system such as T cells and B cells is a
well-documented fact, but such interactions between two different types
of cells of the innate immune system has not been shown previously. The
results described in this work show that the neutrophils and
macrophages interact through CD28-B7, which are known to be the key
regulatory molecules for cytokine production by T cells. We have
identified three major differences between T cell and neutrophil
IFN- secretion. First, while T cells require the first signal
through the Ag-specific TCR to induce the cytokine gene transcription,
the message of which is stabilized by CD28 signaling (33),
neutrophils do not require any primary signal to accomplish the same
function. Absence of such a requirement for the Ag-specific signal in
neutrophil also justifies its Ag nonspecific function during an
inflammatory response. However, it is possible (but remains to be
tested) that neutrophils express a pattern recognition receptor that
signals, as TCRs do in T cells, to induce more IFN-. Second, the
kinetics of IFN- production in neutrophils is different from that of
T cells. While in T cells IFN- production peaks 48 h after
anti-CD3 plus anti-CD28 stimulation, in neutrophils IFN-
production peaks between 6 and 12 h after stimulation. This rapid
production of the cytokine correlates with the observation that the
neutrophils are the first cells to migrate to the site of infection to
encounter foreign materials (1), favoring the age-old
paradigm that the cells of the innate immune system unleash the first
assault on invaders. Third, neutrophils produce significantly less
IFN- than T cells (p < 0.001). A recent
report shows that cytokine levels as low as 50 pg/ml induce Th cell
differentiation (34). This reported level of the cytokine
is equal to the quantity of IFN- that we measured. The observation
implies that, despite low IFN- concentration, the cytokine does not
lose its functional significance, which is amply demonstrated in our
anti-leishmanial assays. Besides the differences in the biological
functions of these two cell types, they also vary in cell surface
costimulatory molecule expression. While T cells express both CD28 and
CTLA-4, which signal in a reasonably counteracting manner in regulating
IL-4 and IFN- secretion from T cells (35), the
neutrophils express only CD28 and not CTLA-4 (data not shown).
When considered from the evolutionary perspective, the marked quantitative and qualitative differences in the cytokine produced by T cells and neutrophils suggest some interesting possibilities. The Ag recognition mechanisms, perhaps the first form of Ag recognition using simple patterns on the Ag, came to existence in phagocytes much earlier than in T cells (36, 37). Therefore, as T cells evolved, the complex MHC-restricted Ag recognition system, which is much finer and detailed, covering wider range of Ags, came into operation (38). Because Igs, MHC molecules, TCR, CD28, and CTLA-4 belong to the Ig superfamily (39), it is possible that CD28 came earlier than the TCR and might have added a finer operational flexibility to the neutrophils. However, phylogenetic studies on these molecules are required to lend further support to this notion.
Because neutrophils are the first cells to encounter
invading pathogen, their primary function is to restrict further
invasion and mobilize a finer course of immune response. It is possible
that a higher amount of IFN- production by neutrophils may lead to
destruction of host tissue. In contrast, a much higher quantity of
IFN- from T cells provides a wider window for finer modulation of
the cytokine production under different conditions. Host tissue
destruction by a high quantity of IFN- may be significantly
inhibited by the counteracting T cell cytokines, such as IL-4 and IL-10
(40, 41), which are not secreted by neutrophils (data not
shown). However, neutrophils do secrete T cell chemotactic factors,
which may draw the T cells to the site of infection, and can modulate
Th subset differentiation (Refs. 5 and 42 and
K. Venuprasad, S. Chattopadhyay, and B. Saha, manuscript in
preparation). Thus, the phylogeny of functions of these two
cells is mirrored during the progress of an Ag-specific immune response
in a two-tiered system. In the first tier, at the beginning of the
response, the neutrophils not only encounter the Ag directly, as
demonstrated using Leishmania infection here, but also lead
to the second tier, where T cells dictate the final outcome of the
infection (42).
In conclusion, neutrophils play a crucial role in the initiation of
anti-leishmanial immune response by secreting IFN- and
restricting parasite growth. The results described in this paper reveal
some novel facts about the differential regulation of T cell and
neutrophil IFN- production that affects the course of
Leishmania infection and perhaps other intracellular
infections as well; they can serve as the basis for further exploration
of the role of neutrophils in Th subset differentiation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bhaskar Saha, National Center for Cell Science, Ganeshkhind, Pune 411007, India. E-mail address: sierra112233{at}hotmail.com
3 Abbreviation used in this paper: PI3-kinase, phosphatidylinositol 3-kinase.
Received for publication February 8, 2002. Accepted for publication May 17, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and chemokines macrophage inflammatory protien-1 and -1 in response to Toxoplasma gondii antigens. J. Immunol. 162:7369. is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils. J. Immunol. 160:5145. T cell repertoire. J. Immunol 160:884.4V8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541.[Medline]
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S. J. Molesworth-Kenyon, J. E. Oakes, and R. N. Lausch A novel role for neutrophils as a source of T cell-recruiting chemokines IP-10 and Mig during the DTH response to HSV-1 antigen J. Leukoc. Biol., April 1, 2005; 77(4): 552 - 559. [Abstract] [Full Text] [PDF]
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; 1:1, 
; 1:5, 
; 1:10, 
; and 1:20, 
) for 2 h. The
extracellular parasites were washed out and the culture supernatants
were collected at different periods of time for IL-8 ELISA.
B, Effect of IL-8 neutralization on the migration of
neutrophils toward macrophages. In a transwell setup, the macrophages
were infected with L. major promastigotes and cultured
for 6 h with or without anti-IL-8 or isotype-matched control
Ab. The neutrophils were put on the upper chamber of the transwell and
the migration assay was performed. Lane A, Medium;
lane B, uninfected macrophages;
lane C, infected macrophages;
lane D, infected macrophages plus
anti-IL-8 Ab; lane E, infected
macrophages plus isotype Ab. C, Effect of IL-8R blockade
on neutrophil migration. In a transwell culture, the macrophages were
infected with L. major promastigotes and cultured for
6 h. The neutrophils were treated with anti-IL-8RA and
anti-IL-8RB Abs and seeded on the transwell insert. The migration
assay was performed as described in Materials and
Methods. Lane A, Untreated neutrophils;
lane B, anti-IL-8RA- and anti-IL-8RB-treated
neutrophils; lane C, control isotype Ab-treated
neutrophils. The results shown are from one representative experiment
that was conducted three times.
