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World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland; and
Pasteur Institute, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Altogether, these observations converged to indicate that events occurring during the first days following infection with L. major are crucial in instructing subsequent Th differentiation. Thus, it is likely that the early transient waves of leukocyte trafficking between the s.c. site of parasite delivery and the draining lymph node could, through the secretion of various factors, influence early events occurring during this period.
Polymorphonuclear leukocytes (PMN)4 are professional phagocytes present only within the blood during steady state conditions. Once the steady state of a peripheral tissue is disrupted, PMN are the first cells recruited within the tissues. Early PMN infiltrate at the site of infection with L. major has been reported, with qualitative and quantitative differences between susceptible BALB/c and resistant C57BL/6 mice. In BALB/c mice, characteristics of an acute inflammatory process, such as persistent elevated numbers of neutrophils, are sustained, whereas this is not the case in mice of resistant strains such as C57BL/6 (12).
Recently, several studies have demonstrated that PMN, once exposed to inflammatory signals, transcribe many genes coding for cytokines and actively synthesize several cytokines, including IL-12 and TGF-ß1 (13, 14, 15). These two cytokines have been reported to influence Ag-specific CD4+ Th cell differentiation. The role of IL-12 in promoting Th1 differentiation has been well established by experiments performed both in vitro and in the murine model of L. major infection (2, 3, 4, 7, 8, 10). The role of TGF-ß in Th differentiation, however, is less clear; depending on the levels present, it may promote either CD4+ Th1 or Th2 differentiation (16, 17, 18, 19).
An immunomodulatory role for PMN has been recently reported in experimental infections with fungi and Toxoplasma gondii where the IL-12 produced by neutrophils was shown to be the initiator of Th1 cell maturation (20, 21). In these models of infection, depletion of PMN correlated with the development of a Th2 response (20) and enhanced mortality (21). These results indicate that, in response to micro-organisms, neutrophils can produce cytokines with immunoregulatory properties that could influence the outcome of the subsequent T cell-dependent immune response. Furthermore, secretion of minute quantities of cytokines by PMN could be relevant due to the elevated number of PMN present within sites of micro-organism inoculation. We therefore investigated whether mAb-mediated depletion of PMN could influence the pathway of Th cell development in mice infected with L. major. The results presented in this report show that depletion of PMN in susceptible (BALB/c) mice infected with L. major hampered the development of a polarized Th2 response. In contrast such treatment did not significantly affect the development of a Th1 response in resistant (C57BL/6) mice. These results suggest that PMN could play an early role in the induction of the Th2 response that develops in BALB/c mice following infection with L. major.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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BALB/c, C57BL/6, and C3H/HeJ mice were obtained from Harlan (Horst, The Netherlands) and housed in pathogen-free facilities at the Epalinges Center.
Parasites, infection, and treatment of mice
L. major (LV 39 MRHO/Sv/59/P strain) were maintained
in vivo and grown in vitro as previously described (22).
Mice were injected in the hind footpads with 3 x
106 stationary promastigotes in a final volume of
50 µl. Affinity protein A column-purified NIMP-R14, a rat IgG2b mAb
that selectively binds to mouse neutrophils (23), was
given i.p. at a dose of 1 mg, 6 h before infection with L.
major. Rat anti human CEA (gift from Dr. J.-P. Mach, Institute of
Biochemistry, Epalinges, Switzerland) was used as control mAb. In some
experiments involving only BALB/c mice an IgG2b mAb against the V
3.2
chain of the TCR (a TCR not present in BALB/c mice) was also used as
control Ab (gift from Dr. R. MacDonald, Ludwig Institute for Cancer
Research, Epalinges, Switzerland). The RB6-8C5 mAb (IgG2b) staining
mouse neutrophils and eosinophils (24) was also used in
some experiments (0.5 mg, administrated i.p. 6 h prior to
infection with L. major).
Treatments with anti IL-12 mAb were performed using a combination of
250 µg of C17.8 and 250 µg of C17.15 mAbs (gift from Dr. G.
Trinchieri, The Wistar Institute, Philadelphia, PA) administered -16,
-4, and 0 h and 2 days following L. major infection.
Treatment with the anti-IFN-
mAb XMG 1.2 (25) was
performed using a single injection of 1 mg of mAb 2 h before
infection with L. major. Following injection with L.
major, lesion progression was monitored using a metric caliper to
quantitate footpad thickness.
Detection of neutrophils within blood and footpads
Neutrophils within the sites of L. major inoculation were detected by microscopic analysis (magnification, x40/high power field) of hematoxylin-eosin-stained sections prepared from paraffin-embedded tissue samples. In selected cases visualization was confirmed by indirect immunostaining with NIMP-R14 mAb followed by streptavidin-PE (PharMingen, San Diego, CA; Becton Dickinson, Mountain View, CA).
PMN depletion following injection of the NIMP-R14 mAb was assessed before and on days 2, 3, and 4 after mAb injection (i.p.) by the analysis of peripheral blood smears stained with DIFFquick (Dade, Düdingen, Switzerland), and/or by FACS analysis, using the biotin-labeled RB6-8C5 mAb staining mouse neutrophils and eosinophils (24) as primary mAb followed by streptavidin-PE or streptavidin-FITC (Becton Dickinson) for PMN detection. Four or 5 h after a single injection of NIMP-R14 mAb the number of circulating neutrophils was dramatically reduced. This treatment led to a transient depletion of PMN, which returned to normal levels in the blood 3–5 days after the injection. It is noteworthy that 21 h following treatment with this mAb, no significant differences were observed in other leukocyte populations within the draining lymph node of mice. The percentage of cells in lymph nodes of mice treated with NIMP-R14 mAb and with the control mAb were as follows: 49 vs 43% of CD4+ T cells, 18 vs 23% of CD8+ T cells, and 33 vs 31% of B cells, respectively.
The numbers of neutrophils within the lesions were estimated using the myeloperoxidase (MPO) assay (26). MPO is a neutrophil enzyme absent in resident tissue macrophages. MPO was measured during the first week following infection with L. major at a time when no increase in footpad thickness was detectable in either resistant or susceptible mice. Briefly, footpads were weighed and homogenized in 1 ml of 100 mM potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide as detergent and 5 mM EDTA. The homogenate was sonicated and centrifuged at 13,000 x g for 15 min. The supernatant (0.1 ml) was diluted 1/15 in the above buffer, and 0.03 ml of 10 mg/ml of a solution of O-dianisidine hydrochloride and 0.001 ml of 0.3% H2O2 was added. The increase in A460 was measured over 2 min. MPO activity is expressed as the change in absorbance units. During the time after infection presently studied, the weights of the footpads of susceptible and resistant mice were equivalent.
Depletion following NIMP-R14 mAb treatment was also assessed in the mouse footpad using this MPO assay (26). Histological analysis of 5-µm sections of footpads stained with hematoxylin/eosin was performed to confirm the results obtained by MPO analysis. The specificity of this treatment in depleting PMN was further assessed by analyzing peripheral blood, the site of L. major injection, and the draining lymph node in mAb treated mice 4 h after infection with L. major. Treatment with the NIMP-R14 mAb resulted in the disappearance of PMN in the three compartments, without modifying the percentage of eosinophils present (Y. Belkaid and G. Milon, unpublished observations).
Detection of cytokines in lymphocyte culture supernatants
Mice infected with L. major were sacrificed at given
times after infection, and 5 x 106 lymph
node cells from the draining popliteal lymph node were restimulated in
vitro in the presence of UV-irradiated L. major
promastigotes in a final volume of 1 ml. Cells were cultured in the
presence of DMEM supplemented with 5% heat-inactivated FCS,
L-glutamine (216 µg/ml), 5 x
10–5 2-ME, and 10 mM HEPES in 7%
CO2. Supernatants were collected after 72 h
of culture. IFN- was measured in supernatant by ELISA as previously
described (27). Mouse rIFN-, used as a standard, was
the supernatant from L1210 cells transfected with the murine IFN-
gene (gift from Dr. Y. Wanabe, Kyoto University, Kyoto, Japan). The
limit of detection is 10 IU/ml. IL-4 was detected by a bioassay using
the CTL.44.A cell line (gift from Dr. P. Erb, University of Basel,
Basel, Switzerland). Recombinant murine IL-4 expressed in X63Ag-653
(gift from F. Melchers, Basel Institute of Immunology, Basel,
Switzerland) was used as a standard. The limit of detection of this
assay is 20 pg/ml. CD4+ T cells were purified
from the draining popliteal lymph node by magnetic cell sorting
(Miltenyi Biotech, Bergish-Gladbach, Germany) according to the
manufacturer’s procedure. Cells (5 x 105)
were stimulated with UV-irradiated promastigotes (1 x
106) in the presence of 5 x
106 irradiated (3000 rad) spleen cells from
normal mice.
RT-PCR
Total RNA was purified from the popliteal lymph node of infected
or uninfected mice, and cDNA synthesis was performed as previously
described (28). The semiquantitative PCR developed by
Reiner et al. with the use of primers for hypoxanthine guanidine
phosphoribosyl transferase, IL-4, and IFN- in the presence of a
polycompetitor was used as previously described (28).
Results are expressed as the fold increase in mRNA transcripts in the
lymph nodes of mice infected with L. major compared with
that in noninfected mice.
The presence of IL-12R ß2 mRNA was monitored using qualitative PCR assay as previously described (11). Briefly, HPRT levels for each sample were assessed by the semiquantitative PCR method of Reiner et al. (28). All samples were normalized with respect to their HPRT content and further subjected to IL-12Rß2 PCR.
Quantitation of parasites
The number of parasites per lesion was evaluated by limiting dilution analysis (29). The estimation of the frequency was calculated by the Taswell method using the program Estimfree (30).
Statistical analysis
Statistical analysis was conducted using the t test for unpaired data.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Before evaluating a potential role for neutrophils during
infection with L. major, we first ascertained whether PMN
were recruited within the site of parasite delivery (footpad). Using
the MPO assay as a correlate to PMN number (26), PMN were
detected in both resistant and susceptible mice as early as 1 h
following injection of 3 x 106 stationary
phase promastigotes. A significant (p < 0.05)
decrease in MPO activity was observed 72 h after infection in
C57BL/6 mice, whereas high MPO activity persisted for up to 6 days
after infection in susceptible BALB/c mice, reflecting the sustained
presence of neutrophils within the site of parasite inoculation in
these mice (Fig. 1
). Administration
6 h prior to infection with L. major of 1 mg of the
PMN-depleting mAb NIMP-R14 prevented PMN accumulation within tissues
following L. major infection, as reflected by a decrease in
MPO activity. The effect of the mAb on the presence of PMN infiltration
16 h postinfection is shown in Fig. 1, right. These
results were confirmed by histological analysis of
hematoxylin-eosin-stained sections of L.
major-infected footpads (data not shown). A significant
difference was thus detected in the neutrophil content of the
infiltrate between mice of susceptible (BALB/c) and resistant (C57BL/6)
phenotypes.
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Groups of BALB/c and C57BL/6 mice were injected i.p. with the
PMN-depleting mAb NIMP-R14 6 h before infection with 3 x
106 L. major stationary phase
promastigotes. BALB/c and C57BL/6 mice injected with a control mAb and
infected similarly were used as control groups. The results in Fig. 2 show that, in contrast to BALB/c mice,
BALB/c mice depleted of neutrophils develop lesions, but only small and
nonprogressing ones at least during the period under study, i.e., 60
days after parasite injection (Fig. 2).
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). Sixty-five days postinfection, while
untreated BALB/c mice had to be sacrificed on day 38 due to the
appearance of necrotic tissues, footpads from NIMP-R14-treated mice
showed only 800 parasites/footpad. The decreased number of parasites
present in footpads of mice treated with the NIMP-R14 mAb probably
reflects enhanced macrophage activation at the site of L.
major inoculation.
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). As complete resolution
of the lesions was also observed in treated mice, the effect of
multiple injections (6 h before and 3 and 6 h after L.
major infection) of the NIMP-R14 mAb was studied. This treatment
was also ineffective in changing the final outcome of infection, and
these mice ultimately resolved their lesions. Depletion of PMN also had
no effect on the final resolution of lesions by mice from another
resistant strain, i.e., C3H/HeJ (data not shown). C57BL/6 mice depleted
in neutrophils showed a 5-fold increase in parasite number within their
lesions 35 days postinfection, but controlled their lesions equally
well at 65 days (Table I). Thus, mAb depletion of PMN resulted in
statistically significant impairment of the development of lesions and
significantly lower parasite load within the infected site in BALB/c
mice. In contrast, such treatment did not alter the final outcome of
infection in either C57BL/6 or C3H resistant mice. Th2 cell maturation is impaired in BALB/c mice depleted in neutrophils before injection with L. major
To evaluate whether the inhibition of lesion progression and
parasite growth in BALB/c mice depleted of neutrophils was correlated
with a shift in the pathway of Th subset differentiation, IFN- and
IL-4 mRNA transcripts as well as protein production were analyzed 10
days after infection, a time when differentiation of the Th subset is
established, in the draining lymph node cells of mice depleted, or not,
of PMN before infection with L. major.
As expected, an increase (24 times) in IL-4 mRNA expression was
observed in control BALB/c mice infected with L. major. Mice
treated with the anti-PMN mAb showed only a 5-fold increase in IL-4
mRNA compared with uninfected NIMP-R14-treated mice, a value considered
not significant with the semiquantitative PCR assay used (Fig. 3A). Levels of IFN-
transcripts were very low and were comparable in both PMN-depleted and
control BALB/c mice, but significantly increased (25 times) in C57BL/6
mice. Injection with the NIMP-R14 mAb resulted in a further increase
(10 times) of IFN- transcripts within the lymph node of
L. major-infected C57BL/6 mice. Comparable results
were obtained with CD4+ T cells isolated from the
draining lymph node; a 15-fold increase in IL-4 mRNA was observed in
CD4+ cells from BALB/c mice, while a 4-fold
increase was observed in NIMPR14-depleted mice 10 days after infection
with L. major.
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3 times) than that of control
infected BALB/c mice (Fig. 3B). Levels of IFN-, however,
were similar (Fig. 3). Draining lymph node cells from C57BL/6 mice
depleted of PMN retained a typical Th1 cytokine profile, with no IL-4
production and an increase (1.6 times) in IFN- proteins (Fig. 3B). Treatment with the RB6–8C5 mAb, an mAb depleting both
neutrophils and eosinophils, given 6 h before infection had a
similar effect on the decreased IL-4 production by
CD4+ T cells (260 vs 6120 pg/ml), while levels of
IFN- were more elevated (2548 vs 638 IU/ml) in RB6–8C5 vs control
treated BALB/c mice, respectively.
A similar analysis was performed 62 days after infection in mice that
had been injected with the NIMPR-14 6 h prior to infection.
Indeed, in this experiment control infected BALB/c mice had to be
sacrificed 32 days postinfection due to the development of necrosing
lesions. L. major-stimulated draining lymph node cells from
BALB/c mice depleted in PMN produced only 800 pg/ml of IL-4, while LNC
from control untreated mice produced 2000 pg/ml of bioactive IL-4 34
days following infection (data not shown). The level of IFN- was
comparable in NIMPP R14-treated or untreated BALB/c and C57BL/6 mice.
Similar differences were noted in a separate experiment in which IL-4
production was measured in both groups of mice 35 days after infection
with L. major (data not shown). These results show that a
single injection of the PMN-depleting NIMP-R14 mAb 6 h prior
infection is sufficient to prevent their subsequent differentiation
along the Th2 pathway normally observed in BALB/c mice infected with
L. major.
Effect of PMN depletion on the early cytokine transcript profile within the draining lymph node of L. major-infected mice
To evaluate whether the presence of PMN could modulate the early
events preceding Th differentiation, BALB/c and C57BL/6 mice were
depleted of PMN by a single injection of the mAb NIMP-R14 followed
6 h later by the s.c. injection of 3 x 106
L. major promastigotes. The levels of IL-4 mRNA in
the draining lymph node were analyzed 16 h following infection and
compared with those in untreated, similarly infected mice. Treatment
with the PMN-depleting mAb prevented the early IL-4 burst of mRNA
transcription normally observed in infected BALB/c mice (Fig. 4). PMN depletion did not induce IL-4
mRNA transcription in C57BL/6 mice (Fig. 4). The early IL-4 mRNA
transcription levels seen in BALB/c mice within 1 day of infection with
L. major have been reported to decrease to values observed
in uninfected mice 48 h postinfection. Treatment with the
anti-PMN mAb did not modify the kinetics of the early IL-4 mRNA
expression or allow its expression in C57BL/6 mice (Fig. 4). Levels of
IFN- mRNA were similar and low in both treated and untreated groups
of BALB/c and C57BL/6 mice at these time points (data not shown). These
results show that a single injection of the NIMP-R14 PMN-depleting mAb
6 h prior infection is sufficient to inhibit the early IL-4 mRNA
burst and the subsequent IL-4 production normally observed in BALB/c
mice infected with L. major.
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The presence of neutrophils within the draining lymph nodes during
the first 72 h following infection with L. major was
assessed by histological examination of hematoxylin-eosin-stained
sections. In BALB/c mice, the presence of PMN within the draining lymph
node was transiently detected, occurring mostly during the first
24 h following infection, with the highest number present 16
h or less postinfection. In C57BL/6-resistant mice, PMN were also
detectable in the subcapsular space at similar time points
postinfection, but in significantly lower number (Fig. 5). These results reveal that PMN are
present transiently in the subcapsular space of draining lymph node
within hours following L. major infection and thereby could
affect processes involved in the early IL-4 mRNA transcription observed
16 h following L. major infection of BALB/c
mice.
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It was previously reported that as soon as 3 days after infection
with L. major, CD4+ T cells from the
draining lymph node of BALB/c mice, unlike those from resistant mice,
lose their capacity to respond to IL-12 in terms of IFN- production
(31). This unresponsiveness was shown to proceed from the
down-regulation of the IL-12 ß2-receptor expression on their
CD4+ T cells (12, 13). To assess the
IL-12 responsiveness of CD4+ T lymphocytes in
PMN-depleted mice, lymph node cells of PMN-depleted or undepleted
BALB/c mice were isolated 5 days after L. major infection
and stimulated with L. major in vitro in the presence or the
absence of rIL-12, and IFN- production was measured. We confirmed
that CD4+ T cells isolated from the draining
lymph nodes of BALB/c mice 5 days after infection with L.
major were unresponsive to IL-12 in terms of IFN- production
(Fig. 6). In contrast, similar to those
from C57BL/6 mice, lymph node cells from BALB/c mice depleted in PMN
exhibited enhanced IFN- production in response to IL-12 (Fig. 6A). Consistently, both C57BL/6 and anti-PMN-treated
BALB/c mice had similar high levels of IL-12Rß2 mRNA in their
draining lymph nodes 5 days postinfection with L.
major (Fig. 6B). Together with results showing an
absence of early IL-4 mRNA burst in PMN-depleted BALB/c mice, these
data indicate that early events occurring in these mice following
infection with L. major correspond to those observed in
resistant mice.
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To monitor whether the shift toward a Th1 pattern following PMN
depletion in BALB/c mice was mediated by IL-12, the effect of the
simultaneous depletion of PMN and neutralization of IL-12 was studied.
Mice were sacrificed 30 days postinfection, and their draining lymph
nodes were isolated for cytokine mRNA analysis. As observed previously,
a single dose of the NIMP-R14 mAb was sufficient to significantly
inhibit the development of lesions. Treatment with mAb against IL-12
abolished the protective effect resulting from PMN depletion (Fig. 7A). Thirty days after
infection with L. major, analysis of draining lymph node
cytokine mRNA revealed high levels of IL-4 mRNA in BALB/c mice treated,
or not, with mAb against IL-12 (40 times more IL-4 than those from
the uninfected, similarly treated mice). Draining lymph nodes of BALB/c
mice depleted of PMN showed very low levels of IL-4 mRNA, not
significantly different from those in mAb-treated uninfected mice.
However, when IL-12 was neutralized by injection of mAb in PMN-depleted
mice, the IL-4 mRNA transcripts were detected at levels comparable to
those in untreated BALB/c mice not depleted in PMN (Fig. 7B). Under these conditions, no IL-4 transcript was detected
in the draining lymph node of C57BL/6 resistant mice as previously
described for mice developing Th1 responses. Accordingly, treatment
with the IL-12 mAb significantly the levels of bioactive IL-4
detected in draining lymph node of cells from BALB/c mice depleted of
PMN (1250 vs 230 pg/ml) as shown in Fig. 7C.
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mAb had a
similar effect, since it also suppressed the decrease in IL-4
production normally observed 10 days after L. major
infection in the draining lymph nodes of mice treated with a single
injection of NIMP-R14 mAb. A nonsignificant 1.8-fold increase in IL-4
mRNA was seen in the draining lymph nodes of anti-PMN mice, whereas
a 40-fold increase in IL-4 mRNA was detected in lymph nodes of mice
simultaneously treated with both IFN and PMN. Furthermore,
measurement of IL-4 in supernatants of specifically stimulated LN cells
substantiated these results by showing 10 times more IL-4 in
supernatants of LN cells of mice treated with both PMN and
IFN- (6154 vs 62 pg/ml). |
Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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In an attempt to assess the role of neutrophils in mice infected with L. major, we have investigated their recruitment within the site of parasite inoculation and their potential role in shaping the L. major-specific T cell response. Profound quantitative differences in the number of PMN recruited were observed between susceptible (BALB/c) and resistant (C57BL/6) mice. Although neutrophils appeared at the site of infection within 1 h in mice from both strains, their number dropped significantly 3 days following infection in resistant mice. In BALB/c mice, their number remained elevated, as estimated by myeloperoxidase activity and histology. These results are in agreement with previous studies using electron microscopic analysis where it was reported that in mice infected with 2 x 107 L. major parasites, the proportion of PMN in the cellular infiltrate in BALB/c mice dropped from 90 to 50% 3 days following infection, persisting for at least 8 days following infection. The percentage of neutrophils in the cellular infiltrate of C57BL/6 mice infected with L. major dropped from 60 to <10% 72 h after infection (12).
The molecular mechanisms underlying the presently observed qualitative and quantitative differences in the number of PMN recruited in the lesions between mice of both strains are not yet deciphered. Differential regulation of adhesion molecules such as VCAM-1/VLA-4 and ß2 integrins/ICAM-1 in susceptible and resistant mice could result in distinct patterns of PMN migration through the endothelial cell of postcapillary venules. Differences in apoptotic rate could also exist between resistant and susceptible strains of mice; mature neutrophils have a short lifespan and constitutively undergo apoptosis. Their lifespan can be increased to days upon recruitment into tissues, presumably due to the actions of particular cytokines. The apoptosis of neutrophil in vitro has indeed been reported to be delayed in the presence of various cytokines, including IL-4 (38, 39). Accordingly, differences in the local cytokine environment between susceptible and resistant mice as a result of infection with L. major could also contribute to the sustained presence of neutrophils within lesions of BALB/c mice and their earlier clearance in C57BL/6. Finally, differences in the maturation of precursor cells in the bone marrow could be involved.
Notable differences have been observed between the ability of PMN from susceptible or resistant mice to destroy intracellular L. major. PMN from BALB/c mice were reported to contain L. major in higher proportion that those from C57BL/6 mice (12). Furthermore, during the first 2 days following infection, parasites within the PMN of BALB/c mice were reported to be intact. In contrast, very low numbers of intact L. major were noted in PMN of C57BL/6 (12) (Y. Belkaid and G. Milon, unpublished observations).
A recent study showed increased numbers of L. major in the footpads of C57BL/6 mice treated with a mAb that depleted both PMN and eosinophils (40). In contrast, no decrease in parasite number was seen in similarly treated BALB/c mice that developed lesions similar to those in control untreated mice. These results differ from those presented here and may be accounted for by the different strains of parasite used, the important amounts of mAb injected repeatedly in their study (a total of 6 mg given 3 and 0 days before and 24 h after L. major infection), and the different spectra of cells recognized by both mAb. In the study byLima et al. the number of PMN in the peripheral blood from untreated BALB/c mice was quite elevated (13.2 ± 1.2 x 106 vs 1.2 ± 0.6 x 106 in the BALB/c mice used in our study). The observed decrease in IL-4 we also observed 10 days after infection with L. major in mice treated with a single dose of the RB6–8C5 mAb 6 h before infection suggests that the differences observed may not be due to the different spectra of cells recognized by both mAb. Furthermore, it has been reported that the number of eosinophils is very low in the lesions of BALB/c mice during the first 3 days after infection (5). Thus, the regimen of mAb might at least in part explain the differences in the results obtained.
We also analyzed the number of neutrophils present within the draining popliteal lymph nodes of infected mice during the first 3 days following inoculation of L. major. Histological examination of paraffin-embedded sections revealed in BALB/c mice a transient increase in the number of neutrophils during the first 24 h after L. major infection. PMN were also detectable in C57BL/6 mice with similar kinetics but in lower numbers. Most neutrophils were located in the subcapsular zone of popliteal lymph nodes of BALB/c mice. The presence of PMN within the lymph node of L. major-infected mice very early following infection indirectly suggests that these cells may contribute to the early events that can influence the pathway of Th cell differentiation. The observation mentioned above that PMN within the lesions of BALB/c mice contain poorly degraded or intact Leishmania raises the possibility that these parasites or parasite fragments could be transported by neutrophils into the lymph nodes, where they could play a role in Ag presentation. Previous studies have shown that human neutrophils can be induced to express MHC class II molecules in vitro (41, 42), and it has been reported recently that human neutrophils can be driven to acquire dendritic cell characteristics in vitro (43). Although in humans PMN have been reported in one study to present superantigens, they did not present classical Ags (42). Thus, the possibility exists that PMN within the lymph nodes of mice infected with L. major could play a role in early Ag presentation either by delivering immunogenic molecules to dendritic cells otherwise known to take up apoptotic cells or, less likely, by presenting the Ags themselves, an issue that deserves further experimental testing.
A role for cytokines secreted by PMN in Th cell differentiation has been reported in several models of infection. The IL-12 secreted by neutrophils has been reported to be associated with the development of a protective Th1 response during infections with Candida albicans and Toxoplasma gondii, whereas secretion of IL-10 by neutrophils was associated with the maturation of deleterious Th2 cells (20, 32, 36, 44). We showed here that depletion of neutrophils before infection with L. major significantly modified the development of lesions and the type of Th response developing in BALB/c mice. The mAb NIMP-R14, which appears to specifically target PMN, has also been reported to prevent both the local and systemic toxicity of LPS due at least in part to the activity of PMN (45).
Prior studies from this laboratory have shown that lymph node cells
from BALB/c mice, in contrast to lymph node cells from mice of
resistant strains, exhibited a burst of IL-4 mRNA transcripts in
CD4+ T cells from the draining lymph nodes within
1 day after infection with L. major (46). This
early burst of IL-4 expression occurred in a restricted population of
Vß4V8 CD4+ T cells after cognate recognition
of a single epitope of the Leishmania homologue of mammalian
RACK1 (47) designated LACK. The IL-4 produced rapidly by
these cells has further been shown to set in motion the molecular
events, including prevention of the IL-12R ß2-chain expression in
CD4+ T cells, ultimately resulting in Th2 cell
maturation and susceptibility to infection in BALB/c mice (11, 47). The results reported here, showing that depletion of PMN in
BALB/c mice hampers the expression of the early IL-4 burst normally
occurring in Vß4V8 CD4+ T cells within 1 day
of infection with L. major, strongly suggest that PMN
contribute to the early events instructing Th2 development in these
mice. The maintenance of the IL-12R ß2-chain expression and the
responsiveness to IL-12 in CD4+ cells from
PMN-depleted BALB/c mice further strengthen the crucial role of the
IL-4 normally produced in these mice during the initial phase of
infection in subsequent Th2 cell maturation.
The precise mechanism(s) accounting for the immunomodulatory role of PMN observed in BALB/c mice infected with L. major are not known. It has been suggested that the function of the IL-12 p40 produced in BALB/c mice early after infection was inhibited by other cytokines produced simultaneously (48). Therefore, it is possible that PMN participate in the production of these cytokines. This hypothesis is supported by previous observations that IL-12 is capable of down-regulating the early IL-4 production seen in BALB/c mice within 1 day of infection with L. major (31). The absence of the early burst in BALB/c mice depleted of PMN could indeed allow expression of the functional activity of IL-12 in absence of inhibitory cytokines.
Two cytokines, IL-10 and TGF-ß, that are secreted by murine neutrophils could potentially play such an inhibitory role. Both of these cytokines have been reported to counteract IL-12-mediated effects in Th differentiation. During infections with L. amazonensis, the production of endogenous TGF-ß correlates with the development of a Th2 response and its associated susceptibility to infection. Less virulent disease associated with the development of a Th1 pattern occurs in animals treated with an mAb against TGF-ß, while more virulent disease occurs in animals given rTGF-ß (49). Furthermore, TGF-ß has been shown to inhibit the IL-12-induced Th1 development of naive CD4+ T cells in vitro (16). Interestingly low concentrations of TGF-ß during primary activation of CD4+T cells inhibit their IL-12 responsiveness (19). Depleting PMN in BALB/c mice could therefore reduce local TGF-ß concentrations and consequently allow the development and sustained responsiveness to IL-12 in CD4+ T cells. The role of neutrophils and their secreted TGF-ß in L. major infections is currently being investigated. IL-10 has also been shown to inhibit IL-12 production during primary Ag response, preventing the development of Th1 responses (50).
PMN have been shown to be involved in the polarization of Th2 immune responses in another experimental model involving artificially induced neutrophilia using G-CSF. Furthermore, in a model of acute graft-vs-host disease the longer survival of recipient of allogeneic cells from G-CSF-treated donors was attributed to the development of a Th2 response, preventing acute graft-vs-host disease (51).
We also show here that in resistant C57BL/6 mice, neutrophils present during the first 3 days of infection do not participate to the development of a Th1 response. These results differ from those obtained in mice infected with C. albicans, in which it was shown that neutrophils, through IL-12 secretion, participate in Th1 cell differentiation (32). Interestingly, bone marrow-derived PMN and L. major-elicited peritoneal PMN obtained from C57BL/6 mice did not show either an increase in IL-12 p40 mRNA or significant IL-12 production following incubation with L. major in vitro (C. Zweifel, J. A. Louis, and F. Tacchini-Cottier, unpublished observations).
In conclusion, the present study shows that neutrophils are recruited early during the first days following infection with L. major in lesions of both resistant and susceptible mice, with a sustained presence only within lesions of susceptible mice. The results presently reported strongly suggest that neutrophils contribute to the development of lesions in susceptible BALB/c mice. Thus, it appears that neutrophils, in addition to contributing to early containment of microorganisms, could also account at least in part for the influence that the innate immune system exerts on the development of adaptive effector T cell responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Fabienne Tacchini-Cottier, World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, 155 chemin des Boveresses, CH-1066 Epalinges, Switzerland.
3 Current address: Pasteur Institute, Cayenne, French Guyane.
4 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; MPO, myeloperoxidase; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
Received for publication February 10, 2000. Accepted for publication June 16, 2000.
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