By
From the * Department of Medicine, Cornell University Medical College, New York 10021;
and Department of Immunology, DNAX Research Institute of Molecular and Cellular
Biology, Palo Alto, California 94304
Although implicated in the clinical expression of human visceral leishmaniasis, a disease-exacerbating T helper cell 2 (Th2)-associated immune response involving interleukin-4 (IL-4) and/
or IL-10 is not readily detectable in experimental visceral infection. To overcome this obstacle
to analyzing visceral Leishmania donovani in this relevant immunopathogenetic environment,
we sought a model in which a Th2 response induces noncuring infection. Four initial approaches were tested primarily in BALB/c mice which control intracellular L. donovani via an
IL-12- and interferon- (IFN-
)-dependent Th1 mechanism: (a) modifying the cytokine milieu when the parasite is first encountered (treatment with exogenous IL-4 or anti-IL-12), (b)
providing sustained endogenous exposure to a Th2 cytokine (infection of IL-4 transgenic
mice), (c) increasing the parasite challenge inoculum, and (d) injecting heat-killed L. major promastigotes (HKLMP) to induce a cross-reactive Th2 response to live L. donovani. Only the last
approach generated a functional Th2-type response that induced disease exacerbation accompanied by inhibition of tissue granuloma assembly. In HKLMP-primed BALB/c mice, prophylaxis with anti-IL-4, anti-IL-10, or exogenous IL-12 (but not IFN-
) readily restored resistance. In primed mice with established visceral infection, treatment with either IL-12 or IFN-
also successfully induced antileishmanial activity. The results in this model (a) suggest that
rather than acting alone, IL-4 and IL-10 may act in concert to prevent acquisition of resistance
to L. donovani, (b) reemphasize the capacity of IL-12 to reverse early Th2-related effects, and (c)
demonstrate that Th1 cytokines (IL-12, IFN-
) have therapeutic action in an established systemic infection despite the presence of a disease-exacerbating Th2-type response.
Emerging data derived from patients with visceral leishmaniasis, a disseminated intracellular protozoal infection
primarily caused by Leishmania donovani or Leishmania chagasi, suggest that clinically apparent or progressive disease
may be related to preferential expression of a downregulating Th2-associated immune response (1). This mechanism, which involves the action of suppressive cytokines
including IL-4, IL-10, and perhaps other soluble factors (12), has been thoroughly characterized in a BALB/c
mouse model of uncontrolled cutaneous infection caused
by Leishmania major (12, 13, 16, 17) and in a distinct model of
cutaneous infection induced by Leishmania mexicana (18, 19).
Although L. donovani and L. chagasi also readily parasitize
and/or cause noncuring visceral infection in inbred mice
(20), these Leishmania species do not regularly provoke
an active, functional Th2 response in experimental infection as they seem to induce in human disease (1, 9).
The one reported exception is in coat pigment mutant
C57BL/6 ep/ep (pale ear) mice in which noncuring L. donovani infection is related to multiple host defense defects including a partially active Th2 cell response (24). However, in other models, it has been difficult to detect or assign a
pathogenic role to this suppressive mechanism. In B10.D2
mice, for example, noncuring L. donovani infection has
been ascribed to the failure to properly express a Th1-associated response rather than to pathologic activity of a Th2
mechanism (23). In addition, although L. donovani does induce both IL-4 and IL-10 expression in infected tissues of
initially susceptible BALB/c mice, this response does not
expand and is rapidly overshadowed by a protective Th1associated mechanism (26). The latter response, dependent
upon IL-12 and IFN- Despite the preceding experience, studying L. donovani
experimentally in the presence of an active Th2 response is
worthwhile in view of the apparent clinical relevance of
this mechanism to human visceral infection (1, 7).
Therefore, we tested a variety of approaches to develop an
L. donovani-Th2 response model.
Mice and Visceral Infection.
20-30 g female BALB/c and 129/
Sv/Ev mice were purchased from Charles Rivers Laboratories
(Wilmington, MA) and Taconic Farms (Germantown, NY), respectively. IL-4 transgenic 129/Sv/PEP mice (30, 31) were bred
at DNAX (Palo Alto, CA). Groups of mice were challenged via
the tail vein with 107 L. donovani amastigotes (1 Sudan strain) obtained from infected hamster spleen homogenates (22). The
course of visceral infection was measured using stained liver imprints, and microscopic counts were performed in a blinded fashion. Liver parasite burdens, expressed as Leishman-Donovan units
(LDU)1, were calculated as the number of amastigotes per 500 nucleated hepatic cells × liver weight (g) (22). Formalin-fixed
liver sections, stained with hematoxylin and eosin, were scored
for granuloma formation as described (22).
Treatment of BALB/c Mice to Induce a Th2-associated Response.
Using the schedules described in the text and figure legends,
groups of four to five BALB/c mice were treated before L. donovani challenge with (a) IL-4 complexed with anti-IL-4 (32) with or without a single injection of anti-IFN- Anticytokine and Cytokine Treatments in HKLMP-primed BALB/c
Mice.
One day before L. donovani challenge (day (27, 28), induces macrophage activation and mediates acquisition of resistance and eventual
resolution of infection (22). The course of visceral L. chagasi
infection in BALB/c mice also closely mimics that of L. donovani, and mice express a susceptible but ultimately selfhealing phenotype (25). In the L. chagasi model, initially
suppressed Th1 activity (IFN-
secretion) is apparently not
related to either IL-4 or IL-10 (25). Finally, rather than being less susceptible to L. donovani, IL-4 gene disrupted 129/ Sv × C57BL/6 mice, in which Th2-associated cytokine
responses are blocked (29), show similar, rather than lower,
parasite burdens in comparison to wild-type controls (19).
, (b) anti-IL-12 (28, 33,
34), or (c) heat-killed L. major promastigotes (HKLPM; 35). 250 µg
of murine recombinant IL-4 (rIL-4; lot 941-90-1; 108 U/ml,
DNAX) was mixed in saline with 2.5 mg of rat anti-murine IL-4
mAb (11B.11; 10 mg/ml; DNAX) (32), and after 2 h on ice, 0.1 ml containing 10 µg of IL-4/100 µg of anti-IL-4 was injected intraperitoneally. This complexing prolongs the in vivo effects of
IL-4 (32). Some IL-4/anti-IL-4-treated mice also received an intraperitoneal injection of 100 µg of anti-murine IFN-
mAb (XMG1.2; 2.6 mg/ml; DNAX). 200 µg of normal sheep IgG or
200 µg of partially-purified sheep anti-mouse IL-12 IgG (5 mg/
ml; Genetics Institute, Cambridge, MA), provided by Dr. V. Van
Cleave (Genetics Institute; 34), was also injected three times per
week intraperitoneally as in earlier studies (28, 34). L. major promastigotes (WHO strain WHOM/
/173), maintained at room
temperature by weekly passage in medium 199 containing 30% fetal bovine serum and antibiotics, were washed once, resuspended in
saline at 1.5 × 108/ml, and then heated at 56°C for 60 min. Mice
were injected subcutaneously (thigh) once per week for 4 wk
with 0.1 ml containing saline alone or 1.5 × 107 HKLMP, and
were challenged with L. donovani 7 d after the fourth injection.
1) and once
a week thereafter, HKLPM-primed mice were injected intraperitoneally with 1 mg of one of the following rat-derived preparations: normal IgG (26), anti-mouse IL-4 mAb (11B.11, lot 3-287880217; Biologic Response Modifiers Program, National Cancer
Institute, Fredrick, MD, provided by Dr. C. Reynolds, National
Cancer Institute; 26), anti-mouse IL-10 mAb (JES5-2A5, IgG1; 2.6 mg/ml; DNAX; 36), or anti-mouse
-galactosidase (GL113,
IgG1; 6 mg/ml, DNAX) used as an isotype control. The latter
two mAb preparations were purified from tissue culture supernatants by ion exchange chromotography and gel filtration, and
each contained <3 endotoxin units/mg of protein (Limulus agglutination assay).
(2 × 105 U/day), or bovine serum
albumin (1 µg/day) as a control was administered continuously
for 7 d by subcutaneously implanted osmotic pumps (Alzet model
2001; Alza Corp., Palo Alto, CA). IL-12 (7.8 × 106 U/mg) and
IFN-
(2 × 107 U/mg) were provided by Dr. J. Sypek (Genetics
Institute) and Amgen Biologicals (Thousand Oaks, CA), respectively. To test cytokine effects as prophylaxis (34, 39), pumps delivering treatment for 7 d were inserted 4 h after L. donovani challenge. To test activity in established visceral infection, 7-d pumps
were implanted 3 wk after infection on day +21. In all groups of
cytokine-treated mice, liver parasite burdens were measured at
the end of week 4 (day +28). Percent inhibition of parasite replication at week 4 was determined as: (week 4 LDU in untreated
mice
week 4 LDU in treated mice) / week 4 LDU in untreated mice × 100; percent parasite killing was determined as:
(week 3 LDU in untreated mice
week 4 LDU in treated mice) /
week 3 LDU in untreated mice × 100 (38).
Assay for Serum IgE.
At the time mice were killed to determine parasite burdens, serum was obtained and frozen at 70°C
until assayed for IgE by ELISA (40).
Statistical Analysis. Differences between mean values were analyzed by a two-tailed Student's t test (24).
Outcome of Visceral Infection in Selected Mouse Populations
Initial efforts were directed at identifying a suitable
model of exacerbated visceral infection that was associated
with induction of a Th2-type response. Fig. 1 summarizes
preliminary experiments in which several different approaches were tested.
Effect of Single-dose Exogenous IL-4.
We focused first on
IL-4, a critical regulator of the Th2 response (12, 13, 19,
41), and tested the effect of altering the host cytokine milieu at the time the parasite was initially encountered.
BALB/c mice were pretreated once the day before infection (day 1) with IL-4 complexed with anti-IL-4 with or
without one injection of anti-IFN-
. 4 wk after a single
dose of IL-4/anti-IL-4, the mean (± SEM) serum level of
IgE (used as a marker for the effect of IL-4; 16, 42) in infected mice was 12.3 ± 1.8 µg/ml (n = 4 mice) versus
4.7 ± 0.4 µg/ml in infected controls (n = 3 mice). As
shown in Fig. 1 A, whereas a single injection IL-4/anti-IL-4 exacerbated infection at week 2, this effect was short-lived
in treated animals. The addition of a single injection of
anti-IFN-
on day
1 did not alter the course of hepatic
infection in IL-4/anti-IL-4-treated mice (n = 5 mice, data
not shown).
The effect of a single
injection of IL-4/anti-IL-4 at week 2, albeit brief, suggested that the continued presence of IL-4 might enhance
the exacerbating action on L. donovani infection. Such an
approach using IL-4 alone transiently promoted L. major infection in resistant mice in one study (42) but not in another (43). Therefore, starting on day 1 and for the first
4 wk thereafter, BALB/c mice were injected twice per
week with IL-4/anti-IL-4. Visceral infection was clearly
promoted during the 4-wk treatment period (Fig. 1 B).
However, once IL-4 injections were discontinued, this effect waned and infection came under control by week 8.
We next turned to IL-4 transgenic mice (30, 31) to test the effect of the sustained presence of endogenous IL-4. Despite a resistant 129/Sv background, these mice develop predominantly Th2-associated responses to L. major and are susceptible to cutaneous infection (31). However, in the face of active secretion of IL-4, as judged by a strikingly elevated serum IgE concentration 4 wk after infection (189 ± 35 µg/ml, n = 5 mice), IL-4 transgenic mice resisted and controlled L. donovani similar to normal 129/Sv/Ev mice (Fig. 1 C). In these latter control animals, serum IgE 4 wk after infection was 13.2 ± 2.0 µg/ml (n = 4 mice).
Effect of Anti-IL-12.IL-12 appears likely to be the key
endogenous initiator of the cell-mediated immune response
which underlies experimental defense against a number of
pathogens including L. donovani (28, 44). Therefore, to explore an alternative approach to establishing a Th2 response, BALB/c mice were treated with anti-IL-12 to inhibit the initiation of the Th1 mechanism, suppress the
principal inducer of endogenous IFN-, and permit cytokines
such as IL-4 and IL-10 to emerge and act unopposed (33,
34, 39). In an initial study of L. donovani-infected BALB/c
mice, three injections of anti-IL-12 during the first week
after challenge were sufficient to largely prevent control of
visceral infection at week 4 (28). In the current experiments, we modified this protocol by (a) injecting BALB/c
mice with anti-IL-12 starting 2 h after L. donovani challenge and then three times per week for 2 wk (rather than
for 1 wk; 28), and then (b) observing the animals for a
longer period after the last anti-IL-12 treatment. Although
anti-IL-12 administration during weeks 1 and 2 exacerbated visceral infection at week 4 (Fig. 1 D), these mice
nonetheless proceeded to control parasite replication and
reduced liver burdens by week 8.
BALB/c mice are remarkably susceptible to L. major and develop unrelenting infection related to induction of a particularly intense Th2 response (12, 13, 45). These events have been linked in part to the size of the original challenge inoculum, because cutaneous infection is controlled when lower numbers of L. major are initially used (46). Therefore, assuming that injecting high numbers of parasites may facilitate induction or enhance the activity of an expressed but nonfunctional Th2 response (26), next we challenged BALB/c animals with 10-20-fold more amastigotes (1 and 2 × 108) than used in the standard L. donovani inoculum (107). However, after four weeks, visceral infection was controlled in a simliar fashion in all three dose groups (Fig. 1 E).
Effect of Pretreatment with HKLMP.Finally, we tested
whether BALB/c mice could be induced by presensitization with four once-per-week injections of HKLMP to cross-react to L. donovani with a Th2-associated response.
This technique provokes a disease-exacerbating effect upon
subsequent infection with live L. major (35). In a preliminary experiment, mice pretreated with four once-per-week
injections of saline alone controlled and eradicated L. donovani; 4 wk after infection, mean (± SEM) liver parasite
burdens were 1,144 ± 86 versus 947 ± 61 in uninjected
controls, and at week 8, were 241 ± 26 versus 260 ± 31, respectively (n = 4 mice per group at each time point). In
contrast, mice pretreated in a similar fashion with HKLMP
failed to express acquired resistance 2 wk after L. donovani
challenge, and liver burdens at week 4 were 3.2-fold higher
than in control mice (Fig. 2). Visceral infection in HKLMPtreated mice then plateaued, and liver parasite burdens remained high at week 8 (8.7-fold greater than in untreated
controls).
Responses in HKLMP-treated BALB/c Mice
Induction of an Active Th2-associated Cytokine Response.In normal unmanipulated BALB/c mice, L. donovani infection induces both IL-4 and IL-10 mRNA expression in the liver and serum IgE levels increase as well (26). Nevertheless, we concluded that this Th2 response is not functional and/or is rapidly overshadowed by a Th1 mechanism in normal mice since treatment with anti-IL-4 (26) or anti- IL-10 (unpublished) does not affect the kinetics of visceral infection and resistance is acquired (22, 26). In contrast, as judged by overt increases in serum IgE levels both before and after L. donovani challenge (Table 1), and more importantly, by the effects of injecting anti-IL-4 and anti-IL-10, HKLMP-primed mice showed clear-cut evidence of an active, disease-promoting Th2-associated cytokine response. As shown in Fig. 3, treatment with either anti-IL-4 or anti-IL-10 permitted HKLMP-sensitized mice to exert essentially normal control over intracellular visceral infection at week 4. Thus, whereas both IL-4 and IL-10 were required, neither by itself appeared sufficient to mediate the suppressive, antihost defense effect of the provoked Th2 response. We concluded from these results that IL-4 and IL-10 likely acted in concert in this model.
|
Effect on the Tissue Immune Response.
Since successful resistance to L. donovani in BALB/c mice is expressed in the
tissues by granuloma formation (22, 47), we also examined
liver sections from HKLMP-pretreated animals. In normal
BALB/c mice, granuloma assembly in the liver proceeds in
an orderly fashion; each infected focus, consisting initially of single, infected resident macrophages (Kupffer cells),
gives rise to a core of fused parasitized Kupffer cells which
comes to be surrounded by a mononuclear cell mantle
comprised of influxing T cells and monocytes (22, 27, 47).
This histologic reaction is detectable by week 2 after challenge and is fully developed by or after week 4 (22, 47).
The early tissue reaction to L. donovani at week 2 was similar in HKLMP-treated and control BALB/c animals: (a) no
cellular reaction was present at 32 ± 6% versus 40 ± 4% of
infected foci, respectively; (b) developing granulomas were
present at 56 ± 8% versus 48 ± 7% of sites; and (c) 12 ± 1% versus 12 ± 2% of foci were scored as mature granulomas (two experiments, n = 4 mice). However, after week
2, responses diverged. While normal mice converted the
bulk of infected foci to mature granulomas by week 8, HKLMP-primed mice did not, and in these animals the tissue reaction failed to properly progress to yield formed
granulomas at the majority of infected sites (Fig. 4). In normal BALB/c mice, single parasitized Kupffer cells with no
surrounding mononuclear cell reaction were seldom encountered 4 (3 ± 1%) or 8 wk (1 ± 1%) after infection
(two experiments, n = 4 mice). Thus, observing little or no
histologic reaction at these latter two time points at 23 ± 4% and 29 ± 5% of parasitized foci in HKLMP-primed
mice also demonstrated the extent of inhibition of mononuclear cell recruitment (Fig. 5). Together, these data emphasized the overall failure of granuloma assembly in this
Th2 response environment.
Effect of Prophylactic and Therapeutic Administration of Th1associated Cytokines IL-12 and IFN-
We concluded the
experiments in HKLMP-sensitized mice by asking (a) whether
the induced Th2 response was reversible by early (prophylactic) treatment with IL-12 or IFN-, two Th1-associated cytokines with well-recognized antileishmanial effects (34, 37), and (b) could exogenous IL-12 or IFN-
also induce activity once the Th2 response and visceral infection
were well established. Consistent with earlier results derived from L. major-infected BALB/c mice (34, 39), early
treatment with IL-12 (days +1 to +7) entirely reversed the
inability of HKLMP mice to control visceral infection measured at week 4 (Table 2). However, in the same experiments, prophylactic treatment with IFN-
had considerably less effect. Nevertheless, when given as therapy in HKLMPsensitized mice with firmly established infection, both IL-12
and IFN-
induced antileishmanial activity (Table 2) at a
level comparable to that achieved in parallel studies carried
out in normal BALB/c mice (37, 48). While the effect of
cytokine treatment in Table 2 is expressed as percentage inhibition of parasite replication, both IFN-
and, to a considerably greater extent, IL-12, also induced leishmanicidal
effects. In cytokine-treated HKLMP-primed mice, liver
parasite burdens were reduced by 26% (IFN-
) and 55% (IL-12) at the end of 7 d of cytokine administration (week
3 versus 4 LDU).
|
These results in HKLMP-primed BALB/c mice (a) describe a model for studying visceral L. donovani in a predominant Th2-associated response environment, (b) not
unexpectedly demonstrate that this response inhibits the
capacity to acquire effective antileishmanial resistance measured by tissue parasite burden and granuloma assembly, and (c) suggest that the disease-exacerbating mechanism in
this model requires the participation of both IL-4 and IL-10.
Among other actions, IL-4 is thought to be primarily responsible for expanding the Th2 response (12, 13, 31, 41);
both IL-4 and -10 are capable of suppressing the secretion
and macrophage-activating effects of Th1-associated cytokines including IFN- (4, 12, 13, 31, 41, 49, 50).
While some of our observations, including the efficacy of prophylactic IL-12 (34, 39), mirror lessons already learned from models of infection caused by L. major and L. mexicana (12, 13, 16, 31, 33, 41), two findings differ. First, although endogenous IL-4 clearly played a resistance-inhibiting role in HKLMP-primed BALB/c mice, 129/Sv IL-4 transgenic mice showed no difficulty in controlling visceral L. donovani. We suspect these results indicate that sustained IL-4 secretion by itself may not be sufficient to overcome the innate resistance (20) imparted by the 129/Sv background of the transgenic animals. However, we did not directly measure IL-4 production, nor investigate other cytokine or cellular responses in infected transgenic mice; thus, potentially suboptimal IL-4 production (30), insufficient cofactor (IL-10) secretion, or the remote possibility of a simultaneously enhanced Th1 response also remain as other potential explanations for our observations.
Second, both IL-12- and IFN--induced visceral antileishmanial activity in the face of an established, disease-exacerbating Th2-associated cytokine response. This new finding
contrasts directly with treatment data derived from models
of established L. major cutaneous infection in BALB/c mice
in which neither exogenous IL-12 nor IFN-
achieved antileishmanial activity (34, 39, 51). There are a number of
possible explanations for these differing observations including the pathogens themselves, the site where infection is
introduced, the requirement in the L. donovani model for
prechallenge manipulation (sensitization), the heterogeneity and/or intensity of a naturally-occurring versus a provoked,
cross-reacting type of Th2 response, and the timing and
doses of cytokine treatment used. Nevertheless, experience
in other models has also raised the possibility of overriding
at least some components of an established Th2 response
using exogenous IL-12 (52). In addition, treatment with
IFN-
alone can also induce measureable antileishmanial
activity in patients with visceral leishmaniasis who also express a Th2 response (9, 60).
Finally, it is worth noting that in HKLPM-primed mice,
exogenous IL-12 was considerably more active than IFN-
when used either prophylactically or as treatment. Since this
study did not include analysis of the mechanisms underlying the efficacy of IL-12 or IFN-
in HKLMP-primed mice,
we have not identified which prohost defense pathway was
enhanced or which suppressive pathway was inhibited.
However, because exogenous IL-12 readily induces IFN-
(37), the enhanced effect of IL-12 in established infection (Table 2) suggests that in addition to macrophage activation stimulated by endogenous IFN-
(38), multiple antileishmanial actions may mediate the efficacy of IL-12 in HKLMPtreated mice. These effects might include the capacity of
IL-12 to expand other Th1-related mechanisms, suppress
the deactivating effects of IL-4 and -10, and perhaps enhance the activity of natural killer cells (44).
Address correspondence to Dr. Murray, Department of Medicine, Cornell University Medical College, Box 130, 1300 York Ave., New York, NY 10021.
Received for publication 18 December 1996.
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