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*
Department of Pathobiology, School of Veterinary Medicine, and
Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and control of L. major
infection. In contrast, C57BL/6 mice deficient in CD28 develop a
dominant Th1-type response and heal infection. In this study, we
investigate the effects of a deficiency in both CD40L and CD28
molecules on the immune response and the course of L.
major infection. We compared infection in mice
genetically lacking CD40L (CD40L-/-), CD28
(CD28-/-), or both
(CD40L-/-CD28-/-), and in C57BL/6 mice, all
on a resistant background. Although CD40L-/- mice failed
to control infection, CD28-/- and
CD40L-/-CD28-/- mice, as well as C57BL/6
mice, spontaneously resolved their infections. Healing mice
had reduced numbers of lesion parasites compared with nonhealing
CD40L-/- mice. At wk 9 of infection, we detected similar
levels of IL-4, IFN-, IL-12p40, and IL-12R
2 mRNA in draining
lymph nodes of healing C57BL/6, CD28-/-, and
CD40L-/-CD28-/- mice, whereas
CD40L-/- mice had increased mRNA levels for IL-4 but
reduced levels for IFN-, IL-12p40, and IL-12R2. In a separate
experiment, blocking of the CD40-CD40L pathway using Ab to CD40L led to
an exacerbation of infection in C57BL/6 mice, but had little or no
effect on infection in CD28-/- mice. Together, these
results demonstrate that in the absence of CD28 costimulation,
CD40-CD40L interaction is not required for the development of a
protective Th1-type response. The expression of IL-12p40, IL-12R2,
and IFN- in CD40L-/-CD28-/- mice further
suggests the presence of an additional stimulus capable of regulating
IL-12 and its receptors in absence of CD40-CD40L
interactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2). In contrast,
nonhealing infections, such as those that occur in BALB/c mice, are
characterized by the development of dominant Th2-like response in which
parasite-specific CD4+ cells produce high levels
of IL-4 and other Th2-associated cytokines but little IFN-. In the
past few years, a number of studies have examined how signaling through
costimulatory molecules affects cytokine production and the subsequent
development of resistance to cutaneous leishmaniasis. These studies
have shown that CD40-CD40L interactions appear to be essential for
induction of a protective cell-mediated immunity to both L.
major and the related protozoan parasite, Leishmania
amazonensis. CD40L-/- or
CD40-/- mice on a resistant C57BL/6 x
129J background are markedly impaired in their production of IL-12 and
IFN- and fail to control their infections (3, 4, 5). In
contrast, studies with other intracellular pathogens like
Histoplasma (6), Mycobacterium
(7), and Toxoplasma (8) have shown
that the CD40-CD40L interactions are not essential for initiating a
protective Th1-type response, although in the case of
Toxoplasma, CD40-CD40L costimulation may be required for
limiting chronic brain encephalitis. The role of B7-CD28 costimulation
in cutaneous leishmaniasis has also been extensively examined.
Treatment of BALB/c mice with CTLA4Ig, which blocks interactions
between B7.1/B7.2 on APCs and both CD28 and CTLA-4 expressed on T
cells, will promote healing if given before infection, but will block
healing if administered continually through infection (9).
However, CD28-/- mice bred onto either the
BALB/c or C57BL/6 background do not exhibit altered patterns of
infection or cytokine production following infection with L.
major, suggesting that CD28-B7 interactions are not absolutely
required for the development of Th2- vs Th1-type responses
(10).
In the present study, we further examined the role of costimulation
during infection using mice that lack either CD40L or CD28 molecules
and mice lacking both CD40L and CD28 molecules. Our results show that
CD40L-/-CD28-/- mice,
in contrast to CD40L-/- mice, control
infection. In addition, these double knockout
(DKO)4 mice fail to
exhibit the defects in IFN- production characteristic of infection
in CD40L-/- mice, suggesting that CD40-CD40L
interaction is not the only pathway for inducing IFN- production
during this infection. Furthermore, in a separate set of experiments,
we show that reduction in the magnitude of the response by lowering the
number of parasites used to initiate infection leads to the activation
of a dominant Th1 response in CD40L-/- mice.
These results suggest that parasite dose as well as costimulatory
pathways can influence the pattern of response to L. major
infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c, C57BL/6, and CD40L-/- mice (B6;129S-Tnfsf5tm1Imx) were purchased from The Jackson Laboratory (Bar Harbor, ME). Dr. C. Hunter at the University of Pennsylvania (Philadelphia, PA) provided CD28-/- mice generated on a C57BL/6 background. CD40L-/-CD28-/- DKO mice breeding pairs were a generous gift from Dr. D. Harlan of the National Institute of Diabetes and Digestive and Kidney Diseases/Navy Transplantation and Autoimmunity Branch (Naval Medical Research Center, Bethesda, MD). The original CD40L KO mice (mixed 129J x C57BL/6 background) were obtained from The Jackson Laboratory. The original CD28 KO mice were on the C57BL/6 background. The CD40L-/-CD28-/- mice used in these studies were the F4 generation. The genotype of the knockouts was verified by PCR screening for the wild-type and KO locus, and confirmed by FACS analysis of splenic T cells for the absence of CD28 and CD40L. All mice were 6–10 wk old at the time of infection. L. major (WHO MHOM/IL/80/Friedlin) was maintained in Grace’s insect cell culture medium (Life Technologies, Grand Island, NY) containing 20% FBS, 2 mM L-glutamine, 100 µg of streptomycin, and 100 U of penicillin G sodium per milliliter.
Infections
Mice were inoculated into one hind footpad with either 1 x 106 stationary-phase L. major promastigotes. In experiments where we used low doses of parasites for infection, mice were inoculated with 1 x 103 to 2 x 104 metacyclic promastigotes selected from stationary phase cultures by negative selection using peanut agglutinin (Sigma-Aldrich, St. Louis, MO) as described previously (11). Lesion size was measured with Vernier caliper and expressed as the difference in thickness between the infected and the uninfected contralateral footpads. Parasites were enumerated by a limiting dilution assay as described previously (12). In brief, the homogenates of infected lesions were serially diluted in Grace’s insect culture medium plus 20% FBS and observed 5–7 days later for growth of promastigotes. Parasite numbers are expressed as the negative log10 dilution at which promastigotes growth was observed.
Anti-CD40L mAb treatment protocol
CD28-/- mice and C57BL/6 mice were treated i.p. with anti-CD40L mAb (MR1; TSD Biosciences, Newark, DE) on day 0 (200 µg/mouse) and then on days 5, 10, and 15 (100 µg/mouse) of infection. Control mice were treated with normal rat Ig.
RNase protection assay
Total RNA was isolated from popliteal draining lymph nodes using RNA STAT-60 (Tel-Test, Friendswood, TX) as directed by the manufacturer. mRNA was quantified by RNase protection assay (RPA) using a Riboquant kit (BD PharMingen, San Diego, CA) as directed. A custom probe from BD PharMingen was prepared using [32P]UTP and hybridized to 15 µg of each sample RNA. The protected probe was purified and resolved on 5% denaturing polyacrylamide gel using Ultra Pure Sequagel reagents (National Diagnostics, Atlanta, GA). Dried gels were exposed to a phosphor imaging screen and protected fragments were visualized using a phosphor imager GS-525 Molecular Imager System (Bio-Rad, Richmond, CA).
ELISPOT assay
The number of IL-12p40 secreting cells in lymph node and spleen cell suspensions was determined using an ELISPOT assay as previously described (13). The mAbs C17.8 and biotinylated C15.6 were generously provided by Dr. C. Hunter (University of Pennsylvania). IL-12 secreting cells were determined in cell cultures following overnight stimulation with 50 µg/ml soluble leishmanial Ag (SLA).
Cell culture and ELISA
Single-cell suspensions of spleens were cultured at 5 x
106 cells/ml in DMEM containing 10% FBS, 2 mM
glutamine, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin
sulfate, and 5 x 10-5 M 2-ME in the
presence of 50 µg/ml SLA (prepared as described previously; Ref.
14). Supernatants were collected at 72 h and assayed
for IFN- by ELISA as previously described (15).
rIFN- (generously provided by Dr. P. Scott, University of
Pennsylvania) was used as standard.
Statistical analysis
Statistically significant differences between groups were determined using the unpaired Student t test. Significance was assumed if p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether combined defects in both CD40-CD40L and
CD28-B7 signaling pathways would alter the response of mice to
infection with L. major, we initially compared the course of
infection in
CD40L-/-CD28-/- mice
with that in CD28-/-,
CD40L-/-, and C57BL/6 mice following
inoculation of 1 x 106 stationary-phase
promastigotes into a hind footpad. As can be seen in Fig. 1
, CD40L-/- mice,
in confirmation of previous reports, failed to control
infection, as evidenced by continued expansion of lesion size
through wk 9 of infection. As expected, C57BL/6 wild-type as well as
CD28-/- mice developed small lesions, which
ultimately resolved. However, unexpectedly,
CD40L-/-CD28-/- mice
resolved their infection in a manner similar to that in
CD28-/- and C57BL/6 wild-type mice. When lesion
parasite numbers were determined at wk 9 of infection,
CD40L-/-CD28-/- mice
were found to harbor 
5 log fewer lesion parasites than
CD40L-/- mice, while C57BL/6 and
CD28-/- mice cleared their infections and had
few detectable lesion parasites (Fig. 2).
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To assess the immune response of the various groups of mice during
infection, we analyzed mRNA levels for cytokines and cytokine receptors
by RPA. The mRNA expression levels of IL-12p40, IL-12R1, and
IL-12R2, as well as IFN- and IL-4 in draining lymph nodes were
compared in
CD40L-/-CD28-/-,
C57BL/6, CD28-/-, and
CD40L-/- mice at wk 9 of infection. As can be
seen in Fig. 3, CD40L-/-CD28-/- mice
expressed mRNA levels for IL-12p40, IL-12R2, and IFN- similar to
those expressed by CD28-/- and C57BL/6
mice. Message for IL-4 in
CD40L-/-CD28-/- mice,
as well as in C57BL/6 wild-type and CD28-/-
mice, was not detected. In contrast, lymph nodes for
CD40L-/- had reduced mRNA levels for IFN-,
IL-12p40, and IL-12R2 receptor, whereas the message levels for IL-4
were enhanced compared with those in the other groups of mice. When
compared with uninfected C57BL/6 mice, message levels for IFN-,
IL-12p40, and IL-12R2 in draining lymph nodes were increased 4- to
5-fold during infection with L. major (data not shown).
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5- to 6-fold higher in infected
CD40L-/-CD28-/- and
C57BL/6 mice compared with their respective naive controls (Fig. 4). Similar results were obtained with
spleen cells of these mice (data not shown).
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We next investigated the effect of blockade of the CD40-CD40L
interactions on the outcome of L. major infection in
CD28-/- mice. We injected
CD28-/- mice as well as C57BL/6 mice i.p. with
anti-CD40L mAb on day 0 (200 µg/mouse), and then on days 5, 10,
and 15 (100 µg/mouse) after infection, and monitored lesion size of
these mice for 9 wk. CD28-/- and C57BL/6 mice
were used as controls to compare the course of infection in
anti-CD40L mAb-treated groups. As can be seen in Fig. 5, treatment with anti-CD40L mAb
exacerbated infection in C57BL/6 mice, but not in
CD28-/- mice. At wk 9 of infection,
anti-CD40L mAb-treated CD28-/- mice had a
reduced number of lesion parasites compared with anti-CD40L
mAb-treated C57BL/6 mice (Fig. 6).
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Because the absence of CD28 costimulation could influence the
magnitude of the response to infection, we examined whether a similar
effect would occur in mice inoculated with a lower number of
promastigotes. CD40L-/- mice were inoculated
with 2 x 104 metacyclic promastigotes and
lesion size was monitored for 8 wk. C57BL/6 and BALB/c mice infected
with same dose were used as controls. As can be seen in Fig. 7, BALB/c mice, as expected, failed to
control infection and developed progressive disease. However,
CD40L-/- mice developed small lesions that
ultimately resolved in a manner similar to those in C57BL/6 mice.
Similar results were obtained when mice were infected with 5 x
103 or 1 x 103
parasites (data not shown). Mice were sacrificed at wk 15 of infection
to determine lesion parasite numbers and cytokine production. Lesions
from BALB/c mice, as expected, contained high numbers of parasites,
whereas lesions from CD40L-/- and C57BL/6 mice
were negative for parasites, suggesting that they had completely
cleared their infections (data not shown). Spleen cells from these
CD40L-/- and C57BL/6 mice produced significant
levels of IFN-, whereas little IFN- was produced by cells from
BALB/c mice (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The development of a dominant Th1-type response, in
turn, is dependent on the presence of IL-12 during the initial stage of
T cell activation. Thus, deletion of the IL-12 gene or treatment of
susceptible strains of mice with rIL-12 or resistance strains with
anti-IL-12 Ab can reverse the expected pattern of infection with
L. major (16, 17, 18, 19). Conversely, mice treated
with anti-IL-4 Ab or mice deficient in the gene for IL-4 or IL-4R
are resistant to infection, consistent with a role for IL-4 in
promoting a Th2-type response (20, 21, 22). In BALB/c mice,
IL-4 production during early stages of infection is believed to
down-regulate IL-12R2 chain expression, resulting in progressive
unresponsiveness to IL-12 (23, 24, 25). In addition to
specific treatments which alter levels of IL-4 or IL-12, or nonspecific
treatments such as sublethal irradiation or in vivo administration of
CTLA4Ig or Abs to CD4 or IL-2, can also promote healing and the
activation of a dominant Th1-type response in susceptible BALB/c mice
(9, 26, 27, 28). Such nonspecific treatments may reduce the
initial level of T cell activation, which, in susceptible mice such as
the BALB/c, would result in reduced activation of IL-4-producing
Th2-type cells.
Because costimulatory molecules play an important role in T cell
activation, it is not surprising that they are equally important in
regulating the in vivo response to L. major. Cells from
BALB/c mice treated with an agonistic Ab to CD40 produce increased
amounts of IL-12 and control infection (29). Conversely,
mice genetically deficient in either CD40 or CD40L, or mice treated
with neutralizing Abs to CD40L, have been shown to be highly
susceptible to infection with either L. major and L.
amazonensis (3, 4, 5, 30). Susceptibility has been
linked to decreased levels of IL-12 during infection, suggesting that
CD40-CD40L interaction, a known stimulus for IL-12 production
(31), is a major stimulus for in vivo IL-12 production.
Kamanaka and colleagues (3) have observed significantly
less expression of IL-12p40 mRNA in cells from
CD40-/- mice compared with those cells from
wild-type mice following infection with L. major. Another
study with L. amazonensis reported a 10-fold reduction in
IFN- mRNA in cells from CD40L-/- mice
compared with IFN- mRNA levels from cells from wild-type mice
(5). In the present study, we confirm that cells from
infected, nonhealing CD40L-/- mice express
elevated mRNA levels of IL-4, but little for IL-12 or IFN-. In
addition, message levels for IL-12R2 in these mice were low compared
with those in wild-type mice, which would be consistent with the
development of a Th2-type response. In contrast, we show that mice
lacking both the genes for CD40L and CD28 can control infection and,
importantly, express mRNA levels of IL-12, IL-12R2, and IFN-
comparable to those in healing C57BL/6 mice. Furthermore, using an
ELISPOT assay, we demonstrate that these mice have numbers of
IL-12p40-producing cells during early stages of infection comparable to
those by C57BL/6 mice. In contrast to infection in
CD40L-/- mice, we observed no evidence of
increased IL-4 production in
CD40L-/-CD28-/- mice,
showing that these mice did not mount a demonstrable Th2-type
response.
Further support for the concept that a deficiency in both CD40L and CD28 costimulation promotes resistance comes from our observation that treatment of C57BL/6 wild-type mice, but not CD28-/- mice, with Ab to CD40L led to increased susceptibility to infection as assessed by increased lesion size. Lesions in Ab-treated wild-type mice ultimately started to heal, which is not unexpected, because anti-CD40L Ab was administered during only the first 2 wk of infection. Heinzel and colleagues (30) have shown similar effects of anti-CD40L mAb on the course of L. major infection in C57BL/6 mice. When parasite numbers were assessed at wk 9, Ab-treated wild-type mice still harbored significantly more lesion parasites than Ab-treated CD28-/- mice.
Our findings on Th1-type cytokine production in
CD40L-/-CD28-/- mice
differ from previous observations which suggest that CD40-CD40L
interaction may be required for protective cell-mediated immunity to
L. major. They also suggest that an alternative mechanism
for inducing IL-12 production must exist in these mice. In contrast to
other intracellular pathogens such as Histoplasma,
Mycobacterium, and Toxoplasma (6, 7, 8),
Leishmania is not thought to directly activate IL-12
production by infected macrophages (32). However, recent
studies have shown that dendritic cells infected in vitro with L.
major or L. donovani do produce IL-12 (33, 34). In addition, it has been suggested that a protein from
L. major, termed Leif, can directly stimulate IL-12
production by human monocytes (35). Thus, it is probable
that stimuli other than CD40-CD40L interactions can promote the
production of this critical cytokine. Why IL-12 production is
up-regulated in DKO mice, but not in CD40L-/-
mice, is unclear. However, we have noted that low mRNA levels for both
IFN- and IL-12 can be detected during early stages (wk 4) of
infection in CD40L-/- mice (data not shown),
suggesting that these mice may produce a weak Th1-type response which
is ultimately down-regulated by an emerging Th2-type response.
In contrast to CD40L-/- mice infected with a high parasite dose (1 x 106) which developed nonhealing lesions and harbored high numbers of parasites, CD40L-/- mice infected with a low dose of parasites (2 x 104 or fewer promastigotes) developed small lesions and ultimately resolved infection consistent with their development of a dominant Th1 response. These results suggest that parasite doses and the level of T cell activation during infection are contributing factors influencing whether costimulatory interactions come into play and alter the pattern of response to infection. Our observations with low-dose experiments differ from previous findings in which intradermal inoculation of 100 L. major metacyclics promastigotes into the ear resulted in the development of nonhealing infections in CD40L-/- mice (36). A possible reason for these differing results may be either the different sites of infection or, more likely, the different genetic background of mice used in these two studies. CD40L-/- mice used in our study were on a mixed background (129 x C57BL/6) and back-crossed on C57BL/6 for fewer than five generations, whereas Belkaid and her colleagues (36) used CD40L-/- mice on the C57BL/6 background, which are known to be more susceptible to L. major infection (4).
The mechanism by which a deficiency in CD28-/-
costimulation influences cytokine production in DKO mice is unclear. As
noted above, CD28-/- mice on a resistant
background produce normal levels of Ag-specific IFN- protein in
comparison to wild-type C57BL/6 mice (10). Several studies
suggest that CD8+ cells can be activated and
differentiated into effector cells in the absence of signals delivered
through CD28 (37, 38). In this regard, we tested whether
treatment of DKO mice with anti-CD8 Ab through the first 5 wk of
infection would alter the course of disease, but we noted no
differences in lesion size between treated and untreated mice through
wk 10 of infection, and anti-CD8-treated mice healed in a timely
manner (data not shown). Recent studies have reported that maximal
responsiveness to IL-12 is dependent on stimulation through CD28, which
results in up-regulation of the IL-12R (39, 40). Lindsten
and colleagues (41) have reported that CD28 not only
up-regulates IL-12R expression but also results in increased stability
of IFN- mRNA, which enhances production of IFN- protein. However,
CD28 costimulation has also been implicated in the activation of Th2
cells (42), and an essential role of CD28 in Th2
differentiation has been described previously, although the operative
mechanism is not known (43). Up-regulation in IL-4 mRNA
level in cells from CD40L-/- mice, but not in
those from DKO mice, is consistent with a role for CD28-mediated
costimulation in promoting the dominant Th2 responses during
Leishmania infection in CD40L-/-
mice. Thus, it is possible that increasing production of IL-4, in
association with reduced production of IL-12, contributes to the
inability of these mice to control infection. Because stimulation
through CD40 can enhance the expression of CD80 and CD86 expression
(44, 45), it is also possible that reduced levels of
CD80/CD86 molecules on APCs may contribute to the reduced level of CD28
costimulation required to drive expansion of IL-4-producing T cells in
mice inoculated with a low dose of parasites. In the absence of high
levels of IL-4 production, it is possible that sufficient levels of
IL-12 and IFN- are produced in both DKO and
CD40L-/- mice to compensate for the deficiency
in IL-12 production that would normally be induced following CD40-CD40L
interaction.
In summary, our results reconfirm the central role of IL-12 in resistance to Leishmania infection but suggest that the requirements for IL-12 production may differ depending on the costimulatory pathways available to individual mice. Whether CD40L-/-CD28-/- mice ultimately control infection because they use alternate costimulatory pathways such as TRANCE-TRANCE-R (46) to induce a dominant Th1-type response or because the absence of costimulation hinders the development of a dominant Th2-type response remains to be determined. However, our results suggest that the reduced level of responsiveness in CD28-deficient mice might be a contributing factor governing whether CD40-CD40L interactions are required for the development of resistance. This hypothesis is supported by results from our low-dose experiments that show that a reduction in the magnitude of the response leads to the activation of a dominant Th1 response in the absence of CD40-CD40L interactions.
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Footnotes |
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2 Current address: Pathophysiological Sciences Initial Review Group, Center for Scientific Review, National Institutes of Health, Bethesda, MD 20892.
3 Address correspondence and reprint requests to Dr. Jay P. Farrell, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: farrellj{at}vet.upenn.edu
4 Abbreviations used in this paper: DKO, double knockout; RPA, RNase protection assay; SLA, soluble leishmanial Ag.
Received for publication March 29, 2001. Accepted for publication September 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or interleukin 4 during the resolution or progression of murine leishmaniasis. J. Exp. Med. 169:59.-independent mechanism. J. Exp. Med. 171:115.
-deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection. Infect. Immun. 68:1773.2 chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. J. Immunol. 161:6156.1 and IL-12R2 mRNAs in CD4+ T cells by costimulation with B7-2 molecules. J. Immunol. 160:1638.1 chain expression and interleukin-12 binding by human peripheral blood mononuclear cells. Eur. J. Immunol. 27:147.[Medline]
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