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/CCL3 in Regulation of T Cell-Mediated Immunity to Cryptococcus neoformans Infection1
*
Veterans Affairs Hospital and
Division of Pulmonary and Critical Care Medicine, The University of Michigan Medical School, Ann Arbor, MI 48109; and
Schering Plough Research Institute, Kenilworth, NJ 07033
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-1/CCL3) is a CC
chemokine required for optimal recruitment of leukocytes in response to
cryptococcal Ags. MIP-1 is expressed in the lungs by day 6 post
Cryptococcus neoformans infection and could play a role
in the development of cell-mediated immunity. To address this
possibility, wild-type (MIP-1+/+) mice and MIP-1
knockout (MIP-1-/-) mice were infected intratracheally
with a highly virulent strain of C. neoformans (145A).
MIP-1 message was detected in the lungs on days 3, 7, and 14 in
MIP-1+/+ mice, but it was undetectable in
MIP-1-/- mice. On day 16, MIP-1-/-
mice had a 7-fold increase in C. neoformans burden in
the lungs, but no decrease in pulmonary leukocyte recruitment.
MIP-1+/+ and MIP-1-/- mice had similar
numbers of recruited lymphocytes and monocytes/macrophages. Notably,
MIP-1-/- mice had a significantly greater number of
eosinophils. MIP-1-/- mice had extremely high levels
of serum IgE. This switch of immune response to a T2
phenotype was associated with enhanced IL-4 and IL-13 expression in the
lungs of MIP-1-/- mice compared with MIP-1
+/+ mice. Progression of pulmonary cryptococcosis in the
presence of nonprotective T2 immunity resulted in profound
lung damage in MIP-1-/- mice (eosinophilic crystal
deposition, destruction of lung parenchyma, and pulmonary hemorrhage).
Twelve-week survival was dramatically decreased in
MIP-1-/- mice. These studies, together with our
previous studies, demonstrate that MIP-1 plays a role in both the
afferent (T1/T2 development) and efferent
(T1-mediated leukocyte recruitment) phases of cell-mediated
immunity to C. neoformans. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, and the
CCR2 (8, 9, 10, 11, 12). In contrast, a T2
response results in IL-4 and IL-5 production, chronic infection with
lung eosinophilia, brain dissemination, and death of mice infected with
C. neoformans (10, 12, 13). Thus,
T1 CMI in the lungs during C.
neoformans infection is protective, whereas
T2 CMI is nonprotective.
Macrophage inflammatory protein-1 (MIP-1) is a CC chemokine
(CCL3) (14) that is required for T1
CMI to C. neoformans. MIP-1 is required for optimal
recruitment of leukocytes in response to cryptococci or cryptococcal
Ags (15, 16). We have shown that, in mice infected
intratracheally with C. neoformans, administration of
neutralizing anti-MIP-1 Abs on days 7–13 of infection decreases
the recruitment of mononuclear phagocytes and neutrophils into the lung
and ablates clearance of the pathogen (15). Thus, MIP-1
plays an important role in the efferent (effector) phase CMI to
C. neoformans, but its role in the afferent phase
(development/polarization) of CMI is unknown.
The early expression of MIP-1 could play a role in the development
of T1 immunity to C. neoformans.
MIP-1 is induced during the early (innate) phase of the immune
response to C. neoformans infection (15, 17).
In other systems, MIP-1 was shown to promote chemotaxis of
Th1 but not Th2 cell lines
in vitro (18). MIP-1 can also drive TCR transgenic
Th0 cells to differentiate to
Th1 cells in vitro (19) and can
decrease IL-4 production from cultured Th2-type
lymphocytes stimulated with schistosomal egg Ag (20, 21).
Anti-MIP-1 Abs can inhibit the development of
T1-mediated experimental autoimmune encephalitis
(19, 22), whereas MIP-1 knockout mice are protected
from Coxsackie virus-induced myocarditis and influenza virus-induced
pneumonitis (23). Thus, MIP-1 has the potential to
modulate the development of CMI. It remains to be determined whether
MIP-1 can modulate the development of T1 vs
T2 CMI in vivo, during infections cleared in a T
cell-dependent fashion, such as C. neoformans.
To determine the role of MIP-1 in regulating the afferent phase of
T1/T2 immune responses, we
performed a series of experiments analyzing pulmonary infection with
C. neoformans in MIP-1 knockout mice
(MIP-1-/-). Clinical isolates of C.
neoformans fall into three categories in experimental murine
infections: low virulent, moderately virulent, and highly virulent
(24). We selected one of the most commonly studied highly
virulent strains of C. neoformans (145A), because one of our
goals was to study the effect of MIP-1 on mouse survival. As
previously demonstrated by our laboratory and Kawakami et al., the
highly virulent class of C. neoformans isolates does not
induce early pro-T1 cytokines, such as TNF-,
IL-12, or IFN-, but surprisingly does not induce a
T2 type response in infected animals
(25, 26, 27, 28). In this report, we demonstrate that production
of MIP-1 prevents the switch to a T2 response
during infection with a highly virulent strain of C.
neoformans. Additionally, we demonstrate that the absence of
MIP-1 has a profound effect on survival of infected host.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MIP-1+/+ mice (B6129F2/J; The Jackson
Laboratory, Bar Harbor, ME) and MIP-1-/-
mice, (B6129F2-Scya3tm1Coo; Ref. 23 ;
University of Michigan breeding colony) were housed under specific
pathogen-free conditions in enclosed filter top cages. Clean food and
water were given ad libitum. The mice were handled and maintained using
microisolator techniques with daily veterinarian monitoring. The
MIP-1-/- mice lack a promoter region, as
well as exon 1 and part of exon 2 of MIP-1 gene (23).
Both MIP-1+/+ and
MIP-1-/- lines of mice were B6129F2
background, and large enough n values were used in the
experiments to control for potential variance of responses within this
strain of mice. Mice were 8–16 wk of age at the time of infection, and
there was no age-related difference in the responses of these mice to
C. neoformans infection.
C. neoformans
Highly virulent C. neoformans strain 145A was obtained from the American Type Culture Collection (62070; Manassas, VA) (28). For infection, yeasts were grown to stationary phase (at least 72 h) at 36°C in Sabouraud dextrose broth (1% neopeptone, 2% dextrose; Difco, Detroit, MI) on a shaker. The cultures were then washed in nonpyrogenic saline (Travenol, Deerfield, IL), counted on a hemocytometer, and diluted to 3.3 x 105 yeast cells/ml in sterile nonpyrogenic saline.
Intratracheal inoculation of C. neoformans
Mice were anesthetized by i.p. injection of pentobarbital (0.074 mg/g body weight; Butler, Columbus, OH) and restrained on a small board. A small incision was made through the skin over the trachea, and the underlying tissue was separated. A bent 30-gauge needle (Becton Dickinson, Rutherford, NJ) was attached to a tuberculin syringe (Becton Dickinson) filled with the diluted C. neoformans culture. The needle was inserted into the trachea, and 30 µl of inoculum was dispensed into the lungs (104 yeast cells). The skin was closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma. Aliquots of the inoculum were collected periodically to monitor the number of CFU being delivered.
Preparation of lung leukocytes
The lungs from each mouse were excised, washed in PBS, minced
with scissors, and digested enzymatically for 30 min in 15 ml/lung of
digestion buffer (RPMI 1640, 5% FCS, antibiotics, 1 mg/ml collagenase
(Boehringer Mannheim Biochemical, Chicago, IL), and 30 µg/ml DNase
(Sigma, St. Louis, MO)). The cell suspension and tissue fragments were
further dispersed by drawing up and down through the bore of a 10-ml
syringe, and centrifuged. Erythrocytes in the pellets were lysed by
addition of 3 ml of NH4Cl buffer (0.829%
NH4Cl, 0.1% KHCO3,
0.0372% Na2EDTA, pH 7.4) for 3 min, followed by
a 10-fold excess of RPMI 1640. Cells were resuspended again, and a
second cycle of syringe dispersion and filtration through a sterile
nylon screen (Nitex, Kansas City, MO) were performed. The filtrate was
centrifuged for 25 min at 3000 rpm in presence of 20% Percoll (Sigma)
to separate leukocytes from cell debris and epithelial cells. Leukocyte
pellets were resuspended in 10 ml of complete media and enumerated in a
hemocytometer, upon dilution in trypan blue. The leukocyte recruitment
was calculated using the following formula: recruited leukocytes in
infected mouse = [(total number of leukocyte in infected
mouse) - (mean number of leukocytes in uninfected mice)].
Leukocyte recovery from uninfected MIP-1+/+
and MIP-1-/- mice was 26.1 ± 3.1
x 106 leukocytes (n = 10) and
26.4 ± 2.6 x 106 leukocytes
(n = 10), respectively.
Assessment of leukocyte population
For the differential count of lung cell suspensions, samples
were cytospun (Shandon Cytospin, Pittsburgh, PA) onto glass slides and
stained by a modification of the Diff-Quik whole blood stain
(Diff-Quik; Baxter Scientific, Miami, FL). Samples were
fixed/prestained 2 min in a one-step methanol based Wright-Giemsa stain
(Harleco, EM Diagnostics, Gibbstown, NJ), rinsed in water, and stained
using steps 2 and 3 of the Diff-Quik stain. This modification of the
Diff-Quik stain procedure improves the resolution of eosinophils from
neutrophils in the mouse. A total of 200–400 cells were counted from
randomly chosen, high-power microscope fields for each sample. The
absolute number of a leukocyte subset was calculated by multiplying the
percentage of each subset in an individual sample by the total number
of lung leukocytes in that mouse. Recruitment was calculated as
described in previous paragraph for total leukocytes. Leukocyte
recovery from uninfected MIP-1+/+
(n = 10) was 0.4 ± 0.1 x
106 neutrophils, 0.2 ± 0.2 x
106 eosinophils, 15.1 ± 1.7 x
106 monocytes/macrophages, and 10.4 ± 1.
8 x 106 lymphocytes; whereas from
uninfected MIP-1 -/- (n =
10), 0.7 ± 0.3 x 106 neutrophils,
0.1 ± 0.1 x 106 eosinophils,
14.7 ± 1.1 x 106
monocytes/macrophages, and 11.0 ± 1.9 x
106 lymphocytes.
Flow cytometric analysis of lymphocyte subsets
Leukocytes (5 x 105) were incubated
for 30 min on ice with staining buffer (FA buffer, (Difco), 0.1%
NaN3, 1% FCS). Each sample was incubated with
the following: 0.12 µg of Cy-Chrome-labeled anti-CD45 (30-F11;
PharMingen, San Diego, CA) and either 0.25 µg each of FITC-labeled
anti-CD4 (RM4–5) and PE-labeled anti-CD8 (53-6.7) or 0.25 µg
of FITC-labeled anti-B220 (RA3–6B2). The samples were washed in
staining buffer and fixed in 2.5% neutral buffered formalin. Stained
samples were stored in the dark at 4°C until analyzed by flow
cytometry (Coulter Elite ESP). Samples were gated for CD45-positive
cells and then analyzed for staining by the specific FITC- and
PE-labeled Abs. The absolute number of a lymphocyte subsets (from
percentages) and their recruitment were calculated in each individual
sample using formulas described in previous paragraphs. Lymphocyte
recovery from uninfected MIP-1+/+
(n = 8) was 2.8 ± 0.3 x
106 CD4+, 1.3 ±
0.3 x 106 CD8+, and
4.3 ± 0.3 B220+ cells/lung; whereas from
uninfected MIP-1-/- (n = 6),
2.4 ± 0.4 x 106
CD4+, 1.7 ± 0.4 x
106 CD8+, and 4.9 ±
1.0 B220+ cells/lung.
Assessment of lung C. neoformans burden
A 100-µl sample from each lung cell suspension was collected from lung digests before erythrocyte lysis. Ten-fold serial dilutions were plated on Sabouraud dextrose agar plates in duplicates. After incubation at room temperature for at least 48 h, CFU were counted and expressed as total CFU per lung.
In vivo treatment with anti-CD4 and anti-CD8 and its evaluation
Mice were injected with 300 µg of GK1.5 mAb (anti-CD4) and 300 µg of YTS 169.4 mAb (anti-CD8) rat IgG on day 0 and 100 µg of each mAb on day 9. Dramatic reduction in T cell numbers was confirmed at day 16 by staining for CD4 and CD8 markers and subsequent flow cytometry of lung leukocytes and splenocytes.
Histology
Lungs were fixed by inflation with 1 ml of 10% neutral buffered formalin. After paraffin embedding, 5-µm sections were cut and stained with hematoxylin and eosin, and viewed by light microscopy.
Detection of cytokine mRNA by RT-PCR
Whole lungs were removed, homogenized in TRIzol Reagent (Life
Technologies, Gaithersburg, MD), extracted as outlined in the TRIzol
protocol, and precipitated with isopropanol. The RNA was washed with
70% ethanol, dissolved in nuclease-free H2O, and
quantified by UV spectrophotometry using absorbance at 260 nm. One step
RT-PCR (Promega Access RT-PCR Kits, Madison, WI) was performed on equal
aliquots of RNA following the manufacturer’s protocol. Three 5-fold
dilutions of the RNA product (1, 0.2, and 0.04 µg) were used for the
RT-PCR to control for possible over-amplification of the cDNA in
the samples. The oligonucleotide primers for PCR were as follows:
-actin, 5'-GTG-GGC-CGC-TCT-AGG-CAC-CA-3' (sense);
5'-CTC-AGC-TGT-GGT-GGT-GAA-GC-3' (antisense); Cyclophilin,
5'-CAT-CTG-CAC-TGC-CAA-GAC-TG-3' (sense);
5'-CTG-CAA-TCC-AGC-TAG-GCA-TG-3' (antisense); IL-4,
5'-GGA-GCC-ATA-TCC-ACG-GAT-GCG-AC-3' (sense);
5'-GAA-TCC-AGG-CAT-CGA-AAA-GCC-CG-3' (antisense); IL-13,
5'-GCC-AGC-CCA-CAG-TTC-TAC-AGC-3' (sense);
5'-CGG-TTA-CAG-AGG-CCA-TGC-AAT-ATC-C-3' (antisense); IL-12p40,
5'-CAG-AAG-CTA-ACC-ATC-TCC-TGG-TTT-G-3' (sense);
5'-TCC-GGA-GTA-ATT-TGG-TGC-TTC-ACA-C-3' (antisense); IFN-,
5'-GGC-TGT-TTC-TGG-CTG-TTA-CTG-CCA-CG-3' (sense);
5'-GAC-AAT-CTC-TTC-CCC-ACC-CCG-AAT-CAG-3' (antisense);
MIP-1, 5'-AAG-GTC-TCC-ACC-ACT-GCC-CTT-G-3' (sense);
5'-CTC-AGG-CAT-TCA-GTT-CCA-GGT-C-3' (antisense).
The PCR parameters for -actin and MIP-1, IFN-, and IL-13
were 35 cycles, and the remaining cytokines underwent 40 cycles.
Annealing temperature was as follows: 55°C for -actin and
MIP-1, 58°C for IL-4 and IL-12p40, and 63°C for IFN- and
IL-13. RT-PCR products were electrophoresed, visualized by ethidium
bromide staining, and the sizes of the RT-PCR products confirmed by
comparison with a 100-bp ladder run in parallel on the same gel. RT-PCR
products were transferred from the gels onto Zeta-Probe Blotting
Membrane (Bio-Rad, Hercules, CA) for 
2 h on a Vacuum Blotter
(Model 735; Bio-Rad). DNA was cross-linked to the membranes in an
UV Stratalinker (Model1800; Stratagene, La Jolla, CA).
Specific DNA products were detected via hybridization with internal
probes for DNA products of interest: -actin,
5'-GGG-ACG-ACA-TGG-AGA- AGA-TCT-GG-3'; IL-4,
5'-TGG-TGT-TCT-TCG-TTG-CTG-3'; IL-12p40,
5'-ATG-TTG-TAG-AGG-TGG-ACT-GG-3'; IL-13,
5'-CCT-GGT-CCA-CAC-AGG-GCA-AC-3'; IFN-,
5'-CAG-CGA-CTC-CTT-TTC-CGC-TT-3'; MIP-1,
5'-GTC-AAC-GAT-GAA-TTG-GCG-TGG-AAT-C-3'.
The probes were labeled with digoxinin (DIG Oligonucleotide 3'-end Labeling Kit; Boehringer Mannheim). Subsequently, DIG Luminescent Detection Kit for Nucleic Acids (Boehringer Mannheim) was used according to manufacturer’s protocol. Presence of specific cDNA (chemiluminescence) on the blots was determined via exposure on x-ray film.
Serum IgE detection (ELISA)
To obtain serum, peripheral venous blood was collected from
MIP-1-/- and
MIP-1+/+ mice at week 5 of infection. Serum
was separated via centrifugation, transferred into microtubes, and
frozen until tested. Murine IgE ELISA kit (PharMingen) was used to
quantify IgE in serum samples diluted 1:100. Reactions were performed
on 96-well ELISA plates (Costar, Cambridge, MA) containing both serum
samples, and the murine IgE standard in duplicates. The ODs were read
on a microplate reader (Ultra Micro EL 808; Biotek Instruments, Luton,
U.K.) at 
= 510 nm. The IgE content in each well was estimated
by interpolation of sample OD values with the murine IgE standard by a
four-parameter curve-fitting program. Sensitivity limit for IgE
detection was 10 ng/ml.
In vivo treatment of MIP-1+/+ mice with
anti-MIP-1 antiserum and evaluation of its effects
Rabbit antiserum against MIP-1 (kindly provided by N. Lukacs
and S. Kunkel, University of Michigan) was used to neutralize MIP-1
in MIP-1+/+ mice. On days 0, 3, and 6 of
C. neoformans infection, MIP-1+/+
mice (n = 5) received this antiserum i.p. Control group
of MIP-1+/+ mice (n = 5),
infected at the same time, received nonimmune rabbit serum i.p.
Following the protocol described above of lung leukocytes isolation,
leukocytes were resuspended in complete medium (5 x
106/ml) and cultured in 24-well plates at 37°C
and 5% CO2 without any additional stimulus.
Supernatants were collected after 24 h and assayed for IL-4 using
the IL-4 ELISA kit (OptEIA; PharMingen) and following manufacturer’s
instructions.
Calculations and statistics
Data (mean ± SE) for each experimental group were derived from three experiments and analyzed via two-way ANOVA. For individual comparisons of multiple groups, post hoc test for simple main effects was used to calculate p values. Means with p < 0.05 were considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the lungs in response to pulmonary
C. neoformans infection
To determine whether MIP-1 is induced during the afferent phase
of the immune response to pulmonary infection with a highly virulent
strain of C. neoformans, we analyzed the time course of
MIP-1 expression in the lungs of intratracheally infected
MIP-1+/+ and
MIP-1-/- mice. Induction of MIP-1 mRNA
could be detected by RT-PCR as early as day 3 in
MIP-1+/+ mice. Levels of MIP-1 mRNA
expression further increased along with the progress of infection on
days 7 and 14 (data not shown). MIP-1-/-
mice did not express this chemokine at any of these time points,
consistent with the deletion of the MIP-1 gene (data not shown).
Thus, pulmonary infection with C. neoformans induces
MIP-1 in the lung by day 3, and MIP-1 expression increases
through the first 14 days of infection.
Effect of MIP-1 deletion on pulmonary growth of C.
neoformans postinfection
To determine the role of MIP-1 in the control of pulmonary
C. neoformans growth, we determined the number of C.
neoformans in the lung of MIP-1+/+ and
MIP-1-/- mice at days 7 and 16. There was no
difference in lung CFU on day 7 (Fig. 1
).
However, at day 16, there was a 7-fold higher C. neoformans
lung burden in MIP-1-/- mice in comparison
with MIP-1+/+ mice (Fig. 1). Thus, MIP-1
does not play a role in controlling growth of C. neoformans
during the first week of infection. However, MIP-1 is required to
control the growth of C. neoformans in the lungs during the
later time points.
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deletion on pulmonary leukocyte recruitment
following C. neoformans infection
To determine whether the increase in lung CFU in
MIP-1-/- mice was due to lack of leukocyte
recruitment upon infection, we compared leukocyte recruitment into the
lungs of C. neoformans-infected
MIP-1-/- and
MIP-1+/+ mice. Leukocytes were isolated from
whole lungs by enzymatic dispersion as described in Materials and
Methods. On day 16 postinfection, there was significant leukocyte
recruitment in both MIP-1+/+ and
MIP-1-/- mice (Fig. 2). Thus, the increased burden of
C. neoformans in MIP-1-/- mice is
not due to deficient leukocyte recruitment into the site of infection
(lungs).
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-/- mice
The next objective was to determine whether MIP-1 deletion
caused a change in the types of recruited pulmonary leukocytes. The
numbers of lymphocytes and monocytes/macrophages recruited into the
lungs were identical in both MIP-1+/+ and
MIP-1-/- mice (Fig. 3A). Additional analysis
demonstrated that there were also no differences in the numbers of
recruited CD4+, CD8+, and
B220+ cells in both strains of mice (Fig. 3B). Neutrophil recruitment was also not defective in
MIP-1-/- mice (Fig. 3A). However,
there was a striking increase in eosinophil recruitment in MIP-1
-/- mice (24 x
106 eosinophils in MIP-1
-/- compared with 7.5 x
106 eosinophils in
MIP-1+/+ (Fig. 3A)). Administration
of anti-CD4 and anti-CD8 mAbs to both strains of mice before
infection resulted in severe reduction of CD4+
(90.6 ± 0.7% in MIP-1-/- and
98.7 ± 0.5% in MIP-1+/+ mice) and
CD8+ cells (94.6 ± 1.9% in
MIP-1-/- and 91.2 ± 2.4% in
MIP-1+/+ mice). This treatment virtually
entirely eliminated eosinophil recruitment in both
MIP-1+/+ and
MIP-1-/- mice (Fig. 3C). Thus,
MIP-1 deletion resulted in a T cell-mediated, pulmonary
eosinophilia, suggesting a switch to a T2-type
immune response to C. neoformans in the absence of
MIP-1.
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+/+ and MIP-1-/- mice
The lungs of C. neoformans-infected
MIP-1-/- and
MIP-1+/+ mice were also examined
histologically at weeks 3, 4, and 10. Both strains of mice developed a
vigorous inflammatory response to pulmonary C. neoformans
infection with accompanying formation of cryptococcomas (Fig. 4). However, the cellular composition of
the C. neoformans-containing foci was different between
MIP-1-/- and
MIP-1+/+ mice.
MIP-1+/+ mice had predominantly a mononuclear
cell infiltrate (Fig. 4, C and E). In contrast,
MIP-1-/- mice had predominantly an
eosinophilic infiltrate (Fig. 4, D and F).
Depositions of eosinophilic crystals were also observed within alveolar
macrophages in MIP-1-/- mice by day 28 of
infection (data not shown) and in the extracellular space at later time
points (Fig. 5A). Areas of
crystal deposition were colocalized with the areas of uncontrolled
growth of cryptococci. These areas contained groups of rapidly dividing
cryptococci with extended capsules (visible as light halos around the
yeast cells) that compressed the surrounding pulmonary tissue and
disrupted the alveolar architecture (Fig. 5, A and
B). Pulmonary hemorrhage was observed as evidenced by
extravascular RBC within the pulmonary airspace in
MIP-1-/- mice (Fig. 5B). To
confirm that the blood extravasation was not a postmortem artifact,
macrophages that had phagocytized and degraded RBC (hemosiderophages)
were sought. These cells were identified based on extended
cytoplasm-containing phagocytized RBC ghosts and a characteristic
olive-brown coloration of hemosiderin (Fig. 5C). The
presence of hemosiderophages provides direct evidence of intravital
bleeding into the airspace of MIP-1-/- mice.
Thus, the lungs of C. neoformans-infected
MIP-1-/- mice show morphological features of
a T2 immune response, including pulmonary
eosinophilia and tissue damage.
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deletion on pulmonary IFN-, IL-12p40, IL-4,
and IL-13 expression following C. neoformans infection
To further determine whether the immune reaction to
Cryptococcus infection shifted to a T2
response in MIP-1-/- mice, we analyzed
pulmonary expression of T1 and
T2 cytokines. IL-4 expression was much stronger
in MIP-1-/- mice than in
MIP-1+/+ mice at day 14 of infection (Fig. 6). Expression of IL-13 was also elevated
in MIP-1-/- mice. There was minimal or no
induction of IL-12p40 and IFN- expression on day 14 of infection in
both groups of mice (Fig. 6). The expression pattern of IL-4 and IL-13
but no IFN- or IL-12 indicates the development of a
T2 response in the absence of MIP-1.
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deletion on serum IgE level
To confirm that the enhanced IL-4/IL-13 expression in
MIP-1-/- mice was indicative of a polarized
T2 response, we looked at the levels of
serum IgE. Infected MIP-1+/+ mice had
extremely low levels of serum IgE, which were not significantly
different from the levels in uninfected mice. In contrast,
MIP-1-/- mice developed extremely high
levels of IgE following C. neoformans infection (Fig. 7). This high level of serum IgE in
C. neoformans-infected MIP-1-/-
mice is consistent with a switch to a polarized
T2 response in these mice.
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neutralization on the phenotype of immune
response to C. neoformans
To exclude the possibility that small genetic differences between
both strains of mice (outside of MIP-1 locus) could account for
changes in phenotype of immune response in our model, we examined the
effects of MIP-1 neutralization in MIP-1+/+
mice. Injections of anti-MIP-1 Abs at days 0, 3, and 6 resulted
in increased eosinophil influx into the lung on day 16 of infection
(21.9 ± 5.9% vs 11.2 ± 2.8%, n = 5
animals per group). The Ab-treated group also had elevated IL-4 in
comparison with nonimmune serum-treated controls (43.1 ± 22.8
pg/ml vs 13.2 ± 3.6 pg/ml, n = 5 animals per
group, measured in supernatant of lung leukocytes cultures). Thus, the
phenotype of the immune response to C. neoformans in
MIP-1+/+ mice treated with anti-MIP-1
during the first week of infection resembles that of
MIP-1-/- mice.
Effect of MIP-1 deletion on the survival of C.
neoformans-infected mice
Following pulmonary infection with C. neoformans,
MIP-1-/- mice began to die by week 6
postinfection (Fig. 8). Cumulative
mortality in MIP-1-/- mice at 12 wk
postinfection was nearly 80%, compared with only 10% in
MIP-1+/+ mice. Thus, deletion of the MIP-1
gene had a dramatic effect on the host response resulting in
significantly decreased survival following pulmonary infection with
C. neoformans.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene
causes a switch to a T2 type of immune response
in a pulmonary infection with a highly virulent strain of C.
neoformans. MIP-1-/- mice developed: 1)
eosinophil influx into the lung and subsequent formation of
eosinophilic crystals; 2) increased IL-4 and IL-13 expression; and 3)
highly elevated IgE levels. This T2 response
produced severe destructive lung pathology (including pulmonary
hemorrhage) without controlling the pulmonary infection. This
nonprotective response resulted in mortality of
MIP-1-/- mice. Thus, expression of MIP-1
prevents the development of a T2 type immune
response to C. neoformans, thereby enhancing survival.
The most striking feature of the response in
MIP-1-/- mice was the massive recruitment of
eosinophils into the lungs of infected animals and subsequent
destructive pathology (Fig. 3
A and 4, D and
F). Lung eosinophilia could be induced also in
MIP-1+/+ mice by MIP-1 neutralization
during the first week of infection, indicating in two independent ways
that absence of MIP-1 in this infection model resulted in increased
eosinophil influx into the lung. Eosinophil recruitment was T
cell-dependent because depletion of CD4+ and
CD8+ lymphocytes abolished eosinophil influx into
the lung (Fig. 3C). Eosinophils were present in the lungs
over an extended time and were accompanied by eosinophilic crystal
deposition in the lungs of MIP-1-/- mice
(Fig. 4, D and F, and 5A). Deposition
of eosinophilic crystals and subsequent lung pathology is a
T2-driven process in C.
neoforman-infected mice (12, 13). In pulmonary
cryptococcosis and other pathologies accompanied by eosinophilic
crystal formation, crystal deposition is associated with epithelial
damage and considered as a source of lung injury (13, 29, 30). Uncontrolled growth of C. neoformans can also
result in destruction of pulmonary architecture (25).
Thus, the combination of uncontrolled cryptococcal growth, pulmonary
eosinophilia, and eosinophilic crystal deposition likely produced the
pulmonary hemorrhage and other lung damage observed in
MIP-1-/- mice (Fig. 5, A and
B). These studies demonstrated that deletion of MIP-1
during C. neoformans infection switched the immune response
to a nonprotective T2 type, resulting in
destructive lung pathology.
Deletion of MIP-1 caused a change in pulmonary cytokine levels
(increased IL-4 and IL-13) that were consistent with the change in
cellular/humoral responses during pulmonary C. neoformans
infection. There was strong induction of IL-4/IL-13 and very little
IL-12/IFN- expression in MIP-1 -/- mice
on day 16 of infection (Fig. 6). In contrast, there was minimal
expression of IL-4/IL-13 in MIP-1+/+ mice.
IL-4 and IL-13 are T2-driving cytokines, and IL-13 is
critically important in T2-type effector
responses (31, 32, 33). MIP-1 has been reported to decrease
IL-4 production in vitro (21). Thus, the enhanced
expression of IL-4/IL-13 compared with IL-12/IFN- in the absence of
MIP-1 explains the switch to a T2-type
immunity in MIP-1 -/- mice. These in vivo
results demonstrate a novel role for MIP-1 in the regulation of T
cell-mediated immunity to an infectious disease.
The high level of serum IgE in MIP-1-/- but
not MIP-1+/+ mice further confirms that an
effective switch to a T2 immune response took
place in these mice. IgE is a hallmark of T2 type
responses, and IL-4 drives B cell isotype switching to IgE production
(34). Thus, the high level of serum IgE in infected
MIP-1-/- mice is consistent with the high
expression of IL-4 in these mice.
Our studies demonstrate that the immunoregulatory effect of MIP-1
takes place during the first week of infection. As demonstrated in
previous studies, treatment with anti-MIP-1-neutralizing Abs during
days 7–14 of infection does not result in this switch
(15). MIP-1 expression was detected by day 3
postinfection. Possible afferent cellular sources of MIP-1 include
alveolar macrophages, CD8+ T cells, 
T
cells, NK cells, and lung epithelium (35, 36, 37, 38, 39, 40, 41). The target
cell(s) of afferent phase MIP-1 remain to be determined. MIP-1
could directly affect expansion of T1 over
T2 cells by increasing IFN- or decreasing
IL-4/IL-13 production (20, 21). MIP-1 is more
chemotactic for T1 than T2
cells in vitro (18). However, T cell recruitment was not
diminished in the absence of MIP-1 during C. neoformans
infection, arguing against the theory of differential recruitment. The
in vivo studies presented in this report demonstrate that afferent
phase MIP-1 production can be a key signal in the development of
T1- vs T2-type CMI
responses in vivo.
In summary, our study has established a novel function for MIP-1 in
the immune system during microbial infection. MIP-1 can function as
a chemotaxin during the efferent phase of immune responses (15, 16, 23, 42, 43, 44). Our studies demonstrate that MIP-1 can also
provide an early signal that down-regulates expression of IL-4/IL-13
during infection, thereby preventing the development of a deleterious
T2-type immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gary B. Huffnagle, Pulmonary Division-Internal Medicine, 6301 MSRB III, Box 0642, 1150 West Medical Center Drive, University of Michigan Medical Center, Ann Arbor, MI 48109-0642.
3 Abbreviations used in this paper: CMI, cell-mediated immunity; MIP, macrophage inflammatory protein; T1, type 1; T2, type 2.
Received for publication April 24, 2000. Accepted for publication August 28, 2000.
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J. J. Osterholzer |
± SE; *, p < 0.05 in comparison with
MIP-1
