INFECTION AND IMMUNITY, Apr. 2004, p. 2338–2349
0019-9567/04/$08.00?0 DOI: 10.1128/IAI.72.4.2338–2349.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 4
Cytokine and Inducible Nitric Oxide Synthase mRNA Expression
during Experimental Murine Cryptococcal Meningoencephalitis
Claudia M. L. Maffei,1,2,3,4Laurence F. Mirels,2,3,4Raymond A. Sobel,5,6
Karl V. Clemons,2,3,4* and David A. Stevens2,3,4
Department of Cellular and Molecular Biology, School of Medicine of Ribeira ˜o Preto of the University of Sa ˜o Paulo,
Ribeira ˜o Preto, Sa ˜o Paulo 14049-900 Brazil1; Division of Infectious Diseases, Department of Medicine, Santa
Clara Valley Medical Center,2and California Institute for Medical Research,3San Jose, California 95128;
Division of Infectious Diseases and Geographic Medicine, Department of Medicine,4and
Department of Pathology,5Stanford University School of Medicine, Stanford,
California 94305; and Palo Alto VA Health Care System,
Palo Alto, California 943046
Received 14 July 2003/Returned for modification 8 August 2003/Accepted 12 January 2004
The immune events that take place in the central nervous system (CNS) during cryptococcal infection are
incompletely understood. We used competitive reverse transcription-PCR to delineate the time course of the
local expression of mRNAs encoding a variety of cytokines and inducible nitric oxide synthase (iNOS) during
progressive murine cryptococcal meningoencephalitis and assessed the CNS inflammatory response using
immunohistochemistry. Interleukin 18 (IL-18), transforming growth factor ?1, and IL-12p40mRNAs were
constitutively expressed in the brains of infected and uninfected mice; IL-2 mRNA was not detected at any time.
Increased levels of transcripts corresponding to IL-1?, tumor necrosis factor alpha (TNF-?), and iNOS were
detected as early as day 1 postinfection, with TNF-? rising by ?30-fold and iNOS increasing by ?5-fold by day
7. Each remained at these levels thereafter. IL-4, IL-6, and gamma interferon transcripts were detected on day
5, and IL-1? and IL-10 transcripts were detected beginning on day 7. Once detected, each remained at a
relatively constant level through 28 days of infection. This cytokine profile does not suggest a polarized Th1 or
Th2 response. Immunohistochemistry did not reveal inflammatory infiltrates before day 7, despite the presence
of cryptococci. Intraparenchymal abscesses with inflammatory cells in their peripheries were found beginning
on day 10. The infiltrates were comprised primarily of cells expressing CD4, CD8, or CD11b; low numbers of
cells expressing CD45R/B220 were also present. The persistence of Cryptococcus observed in the CNS may
result from an ineffective immune response, perhaps owing to an insufficient anticryptococcal effector function
of endogenous glial cells resulting from competing pro- and anti-inflammatory cytokines. These data detail the
immune response in the brain and could be important for the future design of specific immunomodulatory
therapies for this important opportunistic infection.
The ubiquitous yeast Cryptococcus neoformans is an oppor-
tunistic pathogen that causes life-threatening disease, predom-
inantly in patients with impaired cell-mediated immunity, such
as those with AIDS. If the host is unable to effectively clear the
primary pulmonary infection, widespread hematogenous dis-
semination may occur. Cryptococcus appears to have a predi-
lection for establishing meningoencephalitis, as also occurs in
murine models. The immunopathogenesis of central nervous
system (CNS) infection remains incompletely understood (10,
36). Previous studies have suggested that phagocytic effector
cells in the brain (e.g., microglia and astrocytes), as well as
cell-mediated immunity (CMI) and cytokine release, all play
roles in brain-specific immune response (1, 11, 17, 23, 32, 40).
Most studies of cryptococcal infection in murine models
have focused on pulmonary infection, in which the cytokines
tumor necrosis factor alpha (TNF-?), interleukin 1? (IL-1?),
IL-12, IL-18, and gamma interferon (IFN-?) are produced
during the first week of C. neoformans infection. These cyto-
kines appear to stimulate protective CMI (23, 28). Alveolar
macrophages are likely the major source of TNF-? and IL-1?
early in pulmonary infection, whereas the cellular sources of
early IL-12 and IL-18 have not been identified. CD4?and
CD8?T cells and NK cells are potential sources of IFN-? early
in the course of infection (23, 24). Cryptococcal virulence fac-
tors may also modulate early signaling molecules of the host
response. For example, the polysaccharide capsule from C.
neoformans decreases TNF-? and IL-1? production by alveolar
macrophages in vitro and induces production of IL-10 (an
anti-inflammatory inhibitor of Th1 immune response), thereby
providing mechanisms by which the capsule may down-regu-
late CMI in lung or brain (2, 52, 53).
Assessment of mRNA levels is a useful surrogate to measure
the expression of numerous biologically important molecules,
particularly when small amounts of these molecules are pro-
duced. Analysis of mRNA using reverse transcription (RT)-
PCR provides a sensitive tool for studying cytokine regulation.
To quantify this assay, competitive PCR, which employs the
simultaneous amplification of a DNA fragment of interest and
of a competitor template of known concentration within the
same PCR, may be used. The competitor DNA contains prim-
er-binding sites identical to those of the target and is presumed
* Corresponding author. Mailing address: Department of Medicine,
Division of Infectious Diseases, Santa Clara Valley Medical Center,
751 South Bascom Ave., San Jose, CA 95128-2699. Phone: (408) 998-
4557. Fax: (408) 998-2723. E-mail: firstname.lastname@example.org.
to undergo amplification with an efficiency equal to that of the
target. The competitor is designed to yield a final PCR product
that is slightly larger or smaller than that of the target, allowing
the subsequent resolution and identification of their respective
products by using standard agarose gel electrophoresis (18, 29,
To further characterize host defense mechanisms during
cerebral cryptococcal infection, we used competitive RT-PCR
to examine the temporal mRNA expression of a variety of
cytokines and inducible nitric oxide synthase (iNOS) during
the course of experimental murine cryptococcal meningoen-
cephalitis. We concurrently analyzed the inflammatory-cell
types involved in the brain (including adherent meninges) re-
sponse by using immunohistochemistry.
MATERIALS AND METHODS
Mice. Four- to 5-week-old male BALB/c mice were obtained from Charles
River Laboratories (Portage, Mich.). The animals were housed three to five per
cage under conventional conditions and provided sterilized food and acidified
water ad libitum. All experiments were performed with the approval of the
Institutional Animal Care and Use Committee of the California Institute for
Medical Research under the guidelines set forth by the Office of Laboratory
Animal Welfare of the National Institutes of Health.
C. neoformans. An encapsulated serotype A strain of C. neoformans
(CDC9759) was used as described previously (12–14, 37). To enhance virulence,
two consecutives passages in mice were made by intravenous infection and
recovery of yeasts from brain tissue. A single encapsulated colony was picked and
inoculated into 5 ml of synthetic amino acid medium fungal broth (21) and
incubated at 35°C for 72 h on a gyratory shaker at 140 rpm. A 100-?l sample of
this culture was inoculated into 5 ml of fresh synthetic amino acid medium fungal
broth and incubated for 48 h (log phase) on a gyratory shaker at 35°C. The yeast
cells were harvested and washed twice with sterile pyrogen-free saline by low-
speed centrifugation (1,000 ? g), counted with a hemacytometer, and diluted in
saline to 1.6 ? 105cells/ml (inoculum). Serial dilutions of this suspension were
plated on Sabouraud dextrose agar, and the resulting colony counts showed an
inoculum viability of 78%.
Intravenous inoculation. Mice were infected by intravenous injection in a
lateral tail vein with 0.25 ml of the inoculum (4 ? 104cryptococcal yeast cells per
mouse). Uninfected control mice were inoculated with 0.25 ml of sterile pyrogen-
Quantification of C. neoformans in organs. The course of infection was deter-
mined on days 1, 3, 5, 7, 10, 15, 21, and 28 postinfection by quantitative plating
of organ homogenates. In brief, five infected mice were euthanatized by CO2
asphyxiation at each time point. The brain and spleen of each mouse were
aseptically removed and disrupted using a mechanical homogenizer (TekMar,
Cincinnati, Ohio) as described previously (12–14, 37). The number of viable C.
neoformans organisms was determined by quantitative plating of serially diluted
homogenates onto Sabouraud dextrose agar plates containing chloramphenicol
(50 mg/liter). The plates were incubated at 35°C for 3 days, and the numbers of
CFU per entire organ were determined.
RNA extraction and tissue immunohistochemistry. Mice were perfused to
remove peripheral blood cells from the brain and spleen. Three infected mice at
each time point and three uninfected mice on days 5 and 28 were deeply
anesthetized by inhalation of methoxyflurane (Metofane; Schering-Plough Ani-
mal Health, Union, N.J.) vapors. The right atrium of the heart was nicked, and
perfusion was done by inserting a 22-gauge needle into the left ventricle, fol-
lowed by slow injection of 25 ml of 10°C phosphate-buffered saline (PBS). The
brain was removed, and the two hemispheres were separated along the medial
plane; the spleen was also removed. The tissues (half brains or entire spleens)
were put into individually marked polypropylene tubes and immediately frozen
in a dry-ice– acetone bath. All tissues were stored at ?80°C for subsequent RNA
extraction. The remaining half brain was put into a disposable base mold, em-
bedded in Tissue-Tek OCT (Sakura Finetek, Inc., Torrance, Calif.), frozen, and
stored at ?80°C for subsequent sectioning and immunohistochemical analysis.
Total RNA was extracted by homogenizing frozen perfused brain or spleen
tissue in 5 or 3 ml of TRIzol reagent (GibcoBRL, Life Technologies, Rockville,
Md.), respectively, using a mechanical homogenizer at room temperature. The
method used was essentially that supplied by the manufacturer. After homoge-
nization, chloroform (1.0 or 0.6 ml, respectively) was added, and the tubes were
mixed vigorously and then centrifuged at 10,000 ? g and 4°C for 20 min. Total
RNA was precipitated from the aqueous phase with an equal volume of isopro-
panol and pelleted by centrifugation at 13,000 ? g for 10 min at room temper-
ature. Pellets containing RNA were washed once with 75% ethanol and resus-
pended in 200 ?l of diethylpyrocarbonate (DEPC)-treated water. The
concentrations and purities of the RNA preparations were determined by spec-
trophotometry using absorbance at 260 and 280 nm. The A260/A280ratio of the
samples was ?1.8. To confirm the integrity of the RNA and to assess possible
DNA contamination, 10 ?g of each total-RNA sample was electrophoresed and
bands were visualized by ethidium bromide staining.
To remove possible traces of genomic DNA not visualized by the electro-
phoresis, 10 ?g of each RNA sample was treated with 10 U of RNase-free DNase
(RQ1; Promega, Madison, Wis.) at 37°C for 30 min according to the manufac-
turer’s instructions. The DNase was subsequently inactivated by incubation at
65°C for 10 min.
RT. Each DNase-treated RNA sample (5 ?g) was reverse transcribed using 1
?l (0.5 ?g) of oligo(dT)12-18primer (GibcoBRL), 1 ?l (200 U) of SuperScript II
reverse transcriptase (GibcoBRL), 1 ?l (40 U) of RNasin (Promega), 5 ?l of 5
mM (each) deoxynucleoside triphosphate (Sigma, St. Louis, Mo.), 5 ?l of 0.1 M
dithiothreitol (GibcoBRL), 10 ?l of 5? enzyme buffer (GibcoBRL), and 22 ?l of
DEPC-treated water (total reaction volume, 50 ?l). Negative controls were
performed using all components but without added reverse transcriptase. Total
RNA from mouse brain or spleen (Ambion Inc., Austin, Tex.) was used for
positive controls and for establishing reaction conditions. RNA samples, water,
and the primer were initially mixed and incubated at 70°C for 8 min, followed by
a quick chilling on ice. The other reagents were added, and the final reaction
mixture was incubated at 42°C for 1 h. The resultant cDNA was stored frozen at
?20°C until it was needed.
PCR. The following description applies only to PCR done in experiments 2
and 3. The amplification mixture for each sample was made to a total volume of
50 ?l. It contained 0.5 ?l (1 ?g/?l) each of a 3? and a 5? gene-specific primer
(Table 1) (Operon, Alameda, Calif.), 0.5 ?l (5 U/?l) of Taq DNA polymerase
(Promega), 2.5 ?l of cDNA, 21 ?l of DEPC-treated water, and 25 ?l of the
corresponding premix tube of FailSafe PCR (Epicentre Technologies, Madison,
Wis.). The PCR conditions for each primer pair were optimized using cDNA
samples prepared from spleen mRNA and the FailSafe PCR buffers. When
possible for a primer pair, the plasmid pPQRS, a gift from R. M. Locksley
(University of California—San Francisco) (44), was amplified as a positive con-
trol. The plasmid contains a polycompetitor insert for IL-2, IL-4, IL-5, IL-10,
IL-12p40, IFN-?, TNF-?, transforming growth factor ?1 (TGF-?1), iNOS, and
hypoxanthine phosphoribosyl transferase (HPRT) arranged so that the products
amplified from the plasmid template by each specific primer pair differ slightly in
size from those amplified from the cellular target cDNA (44). Negative controls
were samples in which (i) the reverse transcriptase was omitted in the RT step to
test for DNA contamination and (ii) Taq polymerase was not added. The PCR
was carried out in a thermal cycler (Gene Mate; Intermountain Scientific) using
a hot start at 65°C for 2 min and cycles of denaturation at 94°C for 1 min,
annealing at 60°C for 1 min, and extension at 72°C for 2 min (repeated for 35
cycles), with a final end extension of 10 min at 72°C for all targets tested.
Preliminary experiments demonstrated that under these conditions the samples
had not yet reached an amplification plateau. Ten microliters of each PCR
mixture was electrophoresed through 1.5% agarose gels, stained with 0.5 ?g of
ethidium bromide per ml, visualized with a UV transilluminator, and photo-
graphed. The sizes of the PCR products were verified by comparison with a
100-bp DNA ladder run in parallel on the same gel. The specificities of the
transcript-derived bands were determined in gels run with negative controls.
Determination of cytokine mRNA concentration. Samples from infected mice
yielding products of the expected size following PCR with specific cytokine or
iNOS primers (IL-4, IL-10, IFN-?, TNF-?, and iNOS), where no detectable
product was found from uninfected control mice, were subjected to quantifica-
tion using competitive PCR analysis as described previously (18, 29, 44, 58).
Cytokines for which a competitor was not available on the pPQRS plasmid or
that were constitutively produced were not further quantified. Briefly, six serial
PCRs were set up in which a constant volume of experimental cDNA was added
to serial fivefold dilutions of linearized pPQRS plasmid, also in a constant
volume. Primers for IL-4, IL-10, IFN-?, TNF-?, or iNOS cDNA were used for
amplification. PCR products were electrophoresed through an agarose gel and
stained with ethidium bromide. The dilution showing the point of equivalence in
staining intensity of the PCR products from competitor (pPQRS) and from the
tissue samples was used to determine the concentration of the mRNA derived
from a particular sample. To control for possible variation in the efficiencies of
the RT step among different experimental samples, HPRT mRNA concentra-
VOL. 72, 2004CYTOKINE AND iNOS EXPRESSION IN MENINGOENCEPHALITIS2339
tions (a “housekeeping” gene, presumed to be expressed at constant amounts)
were also calculated for each sample.
Immunohistochemistry. Direct and indirect immunohistochemistry was per-
formed on acetone-fixed 8-?m-thick cryostat sections of brain (42). Endogenous
peroxides were inhibited, and the nonspecific protein-binding sites were blocked
by incubating sections with 1% bovine serum albumin (Sigma) in PBS plus 0.0001
M sodium azide for 30 min at room temperature in a humidified chamber. The
following rat anti-mouse monoclonal antibodies (BD Pharmingen, San Diego,
Calif.) were used: biotinylated anti-CD4, 0.5-mg/ml stock diluted 1:100; biotin-
ylated anti-CD8, 0.5-mg/ml stock diluted 1:100; biotinylated anti-CD11b, 0.5-
mg/ml stock diluted 1:100; and purified anti-CD45R/B220, 0.5-mg/ml stock di-
luted 1:200. The antibodies (0.2 ml) were added to cover the tissue sections on
the slides, and the slides were incubated for 1 h at room temperature. For CD4,
CD8, and CD11b, peroxidase-conjugated streptavidin (1.0-mg/ml stock diluted
1:500; Jackson ImmunoResearch, West Grove, Pa.) was added and the slides
were incubated for 30 min at room temperature. An indirect method was used
to detect CD45R/B220, adding biotinylated rabbit anti-rat immunoglobulin G
(IgG) (0.5-mg/ml diluted 1:100; Vector Laboratories, Inc., Burlingame,
Calif.) as the secondary antibody. Reactions were developed using the VEC-
TASTAIN ABC detection system (Vector) for 30 min at room temperature.
The reactions were developed using diaminobenzidine nickel concentrate
(Pierce, Rockford, Ill.) diluted 1:10 in stable peroxide substrate buffer as a
chromogen. The slides were washed three times in PBS for 5 min each time
after each binding step, using gentle agitation. Sections were counterstained
with hematoxylin and mounted. For each antibody, sections of murine spleen
were used as a positive control and nonspecific isotype-matched (purified rat
IgG2a and purified rat IgG2b) antibodies were used as negative controls.
Specificity was confirmed by comparing the observed distribution of label with
the anatomically expected distribution of positive cells in the spleen. The
CD4/CD8 ratio was estimated by counting the CD4-labeled cells in five fields
TABLE 1. Sequences of oligonucleotide primers used for PCR amplification of cytokine, iNOS, and HPRT mRNAs and product sizes
predicted for sample cDNA and competitor DNA (pPQRS)
aAll primer sequences are listed as 5? to 3?. The top primer sequence is the forward primer, and the bottom sequence is the reverse primer for each pair. The primer
pair sequences used for IL-2, IL-4, IL-10, and iNOS were derived from reference 44. The forward primer sequences for TNF-?, IFN-?, and HPRT were derived from
reference 44, and the reverse primer sequences for these pairs were provided by E. Wakil, D. B. Corry, and L. Stowring (personal communications). IL-1?, IL-1?, IL-6,
?-actin, IL-12p35, and IL-12p40primer pair sequences were provided by C. A. Hunter (personal communication). Primer pair sequences for IL-18 and TGF-?1 were
derived from reference 28.
2340MAFFEI ET AL.INFECT. IMMUN.
at ?400 magnification in one section and counting labeled cells in five fields
of another serial section stained for CD8.
Statistical analysis. The yeast burden in tissues is presented as log10geometric
mean CFU per organ ? 95% confidence interval (n ? 5 mice per time point).
Quantification of cytokine transcripts is presented as median log10DNA copies
(n ? 3 mice per time point). Statistical analyses were done using the open-access
program GMC (http://www.forp.usp.br/restauradora/gmc/gmc.html). A nonpara-
metric Kruskal-Wallis analysis of variance followed by a test for multiple com-
parisons (22) was used to test for differences in yeast burdens in tissues for the
various times sampled. Levels of cytokine mRNA transcripts in the brain on the
different days sampled were compared in the same way (22). A P value of ?0.05
was considered significant. Spearman’s rank correlation test was used for corre-
lation analysis. A correlation with an r value of ?0.60 at a level of 1% (? ? 0.01)
was considered significant.
Fungal burden. The recovery of CFU from the various sam-
ples from the brain and spleen is presented in Fig. 1. In the
early phase of infection (days 1 and 3), the yeast burdens in the
brain and spleen were similar. By days 5 and 7, the brain CFU
had increased compared to day 1 CFU (P ? 0.01). From day 5
through day 28, brain CFU remained at ?106/organ, whereas
the spleen CFU decreased significantly with time (P ? 0.05 to
0.01). These data demonstrate clearance of the organisms from
the spleen compared to proliferation in the brain. In addition,
these data delineate the temporal severity of infection, allow-
ing comparison with the cytokine and histological responses.
Cytokine profiles. An initial study of BALB/c mice infected
systemically with C. neoformans was done to determine the
feasibility of following mRNA expression for eight cytokines in
the brain (Table 2, experiment 1). The brains from groups of
four infected and two uninfected mice on days 3 and 10 of
infection and from two infected and one uninfected mouse on
day 21 were examined. mRNA transcripts were followed by
semiquantitative RT-PCR methodology as described for stud-
ies of murine toxoplasmosis (27). In this initial experiment,
?-actin was used as the control housekeeping gene, and in
most cases the primer pairs used (27) were different from those
listed in Table 1. IL-1?, IL-12p35, and TGF-?1 were constitu-
tively expressed by infected and uninfected mice on all days.
IL-2 transcripts were not detected on any day in any mouse.
IFN-?, IL-6, and IL-10 were detected in no animals on day 3 of
infection but were found on day 10 and day 21 only in infected
mice (Table 2, experiment 1). No quantification was done
during these studies, which led us to perform the studies de-
Using the information obtained from the initial study, we
designed a more detailed experiment examining earlier time
points, a broader repertoire of cytokines, and quantitation of
some cytokine transcripts to more clearly define the temporal
sequence of expression. We studied the expression of various
cytokines during the 28-day course of cryptococcal meningo-
encephalitis. Two separate infection experiments were per-
formed (Table 2, experiments 2 and 3). In the initial experi-
ment (Table 2, experiment 2), we sought to optimize the RT-
PCR method and to examine the expression of the various
mRNA transcripts. In the second, replicate experiment (Table
2, experiment 3), the optimized methods were used for the
PCR and mRNA expression was quantified by the competitive-
PCR method. Overall, the levels of expression and times of
appearance of the cytokine mRNAs were very similar for each
experiment. After optimization of the PCR conditions for each
cytokine (Table 2, experiment 3), we detected mRNAs for 11
cytokines (IL-1?, IL-1?, IL-4, IL-6, IL-10, IL-12p40, IL-12p35,
IL-18, IFN-?, TNF-?, TGF-?1) and for iNOS, as well as the
constitutively expressed genes for HPRT and ?-actin, in the
brains of infected mice (Table 2, experiment 3). Transcripts
encoding IL-2 in the brain, if present at all, were at levels below
the sensitivity afforded by this assay. However, mRNA for IL-2
was readily demonstrated in control experiments using RNA
isolated from the spleens of the same animals (data not
shown). The reproducibility of the appearance of mRNA tran-
scripts from individual mice at a given time point is demon-
strated in Table 2 and Fig. 2. In addition, cDNA was made
from the RNA derived from each tissue sample for each ani-
mal on three separate occasions. Replicate PCRs using these
different cDNA preparations showed them to give identical
(i) Constitutive cytokines. mRNAs encoding HPRT, IL-
12p40, IL-18, and TGF-?1 were detected at all sampling times
and at similar levels in the brains, regardless of the presence or
absence of infection (Fig. 2); the same was true for ?-actin and
IL-12p35(data not shown). Quantification by competitive PCR
could not be done for two mRNAs because the primer pair
utilized to amplify IL-12p40mRNA has a different sequence
than that used for the IL-12p40insert of pPQRS and IL-18 was
not included in the pPQRS plasmid. Thus, we examined po-
tential differences by densitometry scans of ethidium-stained
Densitometry analysis suggested comparable levels of tran-
scripts in all animals and throughout the time course for each
of these genes (data not shown). These results indicate that
mRNAs encoding IL-12p35,IL-12p40, IL-18, and TGF-?1 are
constitutively expressed in the brain. One possible exception
was observed for IL-12p40, for which the transcript concentra-
tion increased 40% compared to the baseline beginning on day
10 of infection.
FIG. 1. Yeast burdens in brains and spleens of BALB/c mice after
intravenous infection with 4 ? 104cells of C. neoformans (isolate
9759). The data are expressed as log10geometric mean CFU per organ
? 95% confidence interval (n ? 5 mice/time). The differences between
brain and spleen at each time compared to day 1 postinfection were
statistically significant as indicated (*, P ? 0.05; **, P ? 0.01; ***, P ?
VOL. 72, 2004CYTOKINE AND iNOS EXPRESSION IN MENINGOENCEPHALITIS 2341
TABLE 2. Expression of cytokine and iNOS mRNAs during the course of experimental meningoencephalitis due to C. neoformans, analyzed
Cytokine or control Expt no.
Infected miceUninfected mice
357 101521 2835 1028
aRNA samples were obtained from two to four mice per time point. The symbols indicate whether mRNA for a specific gene was detected after PCR amplification
for each animal (i.e., one symbol represents one animal). ND, RT-PCR was not done for that time point; ?, no mRNA was detected for that gene at the lower limit
of sensitivity of the assay; ?, mRNA expression was detected for that gene; ?, mRNA expression was at the limit of detection; #, mRNA expression was detected for
that gene and, as measured by competitive PCR, was at a significantly higher level than mRNA expression for the gene on days 1 and 3 postinfection (P ? 0.05 to 0.001)
(not used for products when quantification was not performed by RT-PCR; those are indicated as mRNA detected only [?]).
(ii) Inducible cytokines. The levels of IL-1?, IL-1?, IL-4,
IL-6, IL-10, IFN-?, TNF-?, and iNOS mRNAs were induced
or elevated in the brain during the course of infection (Fig. 2).
Cytokine mRNAs for IL-1?, IL-4, IL-6, IL-10, and IFN-? were
not detectable in uninfected controls. TNF-? was detected in
one uninfected mouse (Fig. 2, lane 4), but this animal was not
included in further analyses. IL-1? was detected in repeated
amplifications in some controls, but not others, as a barely
visible band and may be expressed on a constitutive basis at
very low levels. In infected animals, these cytokine mRNAs
showed differences in their times of initial appearance postin-
fection. IL-1?, TNF-?, and iNOS were detectable 1 day postin-
fection, whereas IL-4, IL-6, and IFN-? were detected begin-
ning on day 5 and IL-1? and IL-10 were detected beginning on
day 7 postinfection (Fig. 2).
(iii) Cytokine mRNA concentration. The method of compet-
itive PCR used to quantify the number of transcripts is illus-
trated in Fig. 3. For IL-4, there was a significant increase in
transcript abundance on days 7, 15 and 21 (P ? 0.01) and on
day 28 (P ? 0.001) in comparison with the levels at the first
FIG. 2. Temporal expression of cytokine, iNOS, and HPRT mRNAs in brains of uninfected BALB/c mice (lanes 1 to 6) and BALB/c mice
infected with 4 ? 104cells of C. neoformans (lanes b to z). Total RNA was extracted from half of each brain from uninfected mice on days 5 and
28 and from infected mice on days 1, 3, 5, 7, 10, 15, 21, and 28. The RNA samples were obtained from three or four mice per time point. RT-PCR
was performed, and the PCR products were electrophoresed on 1.5% agarose gels containing 0.5 ?g of ethidium bromide per ml and observed
with a UV transilluminator. Lane a, molecular size marker (100-bp ladder). (A) Proinflammatory cytokines; (B) Th2 cytokines; (C) Th1 cytokines;
(D) inducible enzyme; (E) constitutive cytokines; (F) internal control.
VOL. 72, 2004 CYTOKINE AND iNOS EXPRESSION IN MENINGOENCEPHALITIS2343
detectable time point, day 5. However, maximal induction had
occurred by day 7 postinfection and remained constant there-
after (Fig. 4).
The induction of measurable quantities of TNF-? mRNA
began on day 1 and persisted during the entire course of
infection. The numbers of copies (log10) of TNF-? mRNA
were significantly greater on days 7, 10, 15, 21, and 28 than on
day 1 or 3 (P ? 0.01 to 0.001). These levels remained high and
were higher on day 28 postinfection (P ? 0.05 to 0.01) than at
any preceding time point (Fig. 4).
Figure 4 also shows that the results for iNOS were similar to
those for TNF-? (days 7 through 28 compared to day 5; P ?
0.05 to 0.01). After day 5 or 7, there was no significant change
in the levels of mRNA transcripts for IL-4 (day 7), IFN-? (day
5), and IL-10 (day 7) (Table 2 and Fig. 4).
Correlation analysis of the transcript levels of induced genes
that had been quantified was done by the Spearman rank
correlation test. These results showed direct correlation at the
1% level between the expression levels of IL-4 and iNOS (r ?
0.73). No other significant correlations in gene expression were
Inflammatory-cell response in brain. To determine whether
the observed variations in cytokine levels were associated with
particular cell types recruited to the site of the infection, im-
munohistochemical staining for T cells, B cells, and monocytes
was performed on frozen sections of brain obtained at various
times during the course of infection. Small cryptococcal
pseudocysts containing densely packed accumulations of en-
capsulated organisms were observed by day 5 postinfection.
The first inflammatory cells associated with these lesions were
detected on day 7; these cells were CD11b?. Since few neu-
trophils were identified in any of the infiltrates, the CD11b?
cells were probably predominantly macrophages or monocytes
(Table 3). On day 10, acute meningitis and intraparenchymal
FIG. 3. Competitive PCR. (Left panel) Schematic of a typical PCR experiment (described in Materials and Methods). (A) Titration of iNOS
mRNA transcripts in the brains of infected BALB/c mice sampled at different times postinfection. A constant amount of experimental cDNA was
titrated against sequential dilutions of linear pPQRS plasmid competitor during the PCR amplification. Lane 1, 3.8 ? 105copies of plasmid
DNA/?l; lane 2, 7.7 ? 104copies of plasmid DNA/?l; lane 3, 1.5 ? 104copies of plasmid DNA/?l; lane 4, 3.1 ? 103copies of plasmid DNA/?l;
lane 5, 6.2 ? 102copies of plasmid DNA/?l; lane 6, 1.2 ? 102copies of plasmid DNA/?l. The arrowheads indicate the points of equivalence in
copy numbers of the two different PCR products. The sizes of the PCR products were 390 bp for pPQRS iNOS and 306 bp for iNOS. (B) Example
of the reproducibility of the pPQRS methodology for the expression of iNOS transcripts in three mice (a, b, and c) at the same time postinfection.
The lanes are the same as for panel A.
2344 MAFFEI ET AL.INFECT. IMMUN.
abscesses with many encapsulated cryptococci were evident.
The inflammatory cells consisted of CD4?and CD8?T lym-
phocytes, CD11b?cells, and a few CD45R/B220?B lympho-
cytes. The CD4/CD8 ratio was estimated to be 2.5:1. The same
pattern of inflammation was seen on days 10, 15, 21, and 28
postinfection (Fig. 5).
In general, abscesses increased in size with time, and they
were more numerous, containing more thickly encapsulated
FIG. 4. Quantification of DNA copies of cytokines (IFN-?, TNF-?, IL-4, and IL-10), iNOS, and HPRT during the course of cerebral
cryptococcal infection in BALB/c mice, determined by competitive PCR as described in Materials and Methods. The symbols represent individual
mice, and the bars represent the medians for the time points examined. On days 1 and 3, IFN-?, IL-4, and IL-10 were not detected, and IL-10 was
not detected on day 5. The statistical significance of differences during the evolution of infection and the correlation among the cytokines are
discussed in the text. Values of 2 indicate that no product was detected, and the lower limit of sensitivity of the assay was ?100 copies.
VOL. 72, 2004CYTOKINE AND iNOS EXPRESSION IN MENINGOENCEPHALITIS2345
budding yeast. The inflammatory-cell response and the num-
bers of T cells, B cells, and monocytes visualized in the lesions
generally correlated with the numbers of yeast cells present in
the abscesses. No necrosis, vasculitis, granulomatous foci, or
multinucleated or epithelioid cells were observed at any time.
Numerous reports have described the immune interactions
that take place in the lungs following infection with C. neofor-
mans (19, 20, 23, 24, 40, 43). Less is known about the host-
pathogen interplay in the brain, the organ most frequently
involved in extrapulmonary infection and in infections with a
Although the CNS has traditionally been regarded as an
immunologically privileged site, activated T cells can traffic
across the blood-brain barrier into the CNS for immune sur-
veillance. In this way, when subjected to an injury or infection,
the CNS can mobilize and develop an immune response in-
volving infiltrating CD4?and CD8?T cells, B cells, macro-
FIG. 5. (A and B) Cryosections of typical intraparenchymal cryptococcal abscesses from day 10 (A) and day 21 (B) postinfection (hematoxylin
and eosin staining). (C to F) Adjacent serial cryosections (immunoperoxidase with hematoxylin counterstain) demonstrating the inflammatory
response in a cryptococcal abscess from day 28 postinfection. Intact brain parenchyma is on the left side of the field in each panel. (C) Anti-CD4;
(D) anti-CD8; (E) anti-CD11b; (F) anti-CD45R/B220. Numerous CD4?and CD8?lymphoid cells and CD11b?cells are shown in the abscess.
There are fewer B cells in the infiltrate in panel F. Magnifications: ?206 (A), ?171 (B), and ?294 (C to F).
TABLE 3. Presence of T lymphocytes (CD4?and CD8?), B lymphocytes (CD45R/B220?), and monocytes (CD11b?) in the brain during
Presence on daya:
1357 1015 21 28
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
? ? ?*
? ? ?*
? ? ?*
? ? ?*
?* ? ?*
?* ? ?*
?* ? ?*
?* ? ?*
?* ? ?* ?*
?* ? ?* ?*
?* ? ?* ?*
?* ? ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
?* ?* ?*
a?, absence of labeled cells; ?, presence of labeled cells; *, Cryptococcus in tissue. CD4, CD8, and CD11b were detected directly using biotinylated rat anti-mouse
antibodies, and CD45R/B220 was detected indirectly using purified rat anti-mouse antibody followed by biotinylated rabbit anti-rat immunoglobulin G. All were
observed after immunoperoxidase staining. Three or four mice were evaluated at each time point as shown by the number of symbols.
2346 MAFFEI ET AL.INFECT. IMMUN.
phages and neutrophils, and activated resident cells (e.g., mi-
croglia, astrocytes, and endothelial cells) (6, 7, 10, 17, 31, 57).
When they are activated, endogenous CNS cells express
major histocompatibility complex class I and class II molecules
and may therefore act as antigen-presenting cells. They also
express complement receptors; produce cytokines, chemo-
kines, and molecules with bactericidal activity, such as nitric
oxide (NO); and are capable of phagocytosis (1, 7, 10, 25, 26,
30, 32, 57). Microglia, acting as antigen-presenting cells, stim-
ulate T-cell proliferation and cytokine secretion, which in turn
stimulates these semiprofessional phagocytes to ingest and
more effectively kill invading organisms (1, 57).
Whether an immune response within the CNS is initiated,
amplified, or suppressed depends on a number of factors, in-
cluding (i) the activation state of microglia, (ii) cytokine and
cytokine receptor levels in glial and immune cells, (iii) relative
expression of immune-enhancing and immune-suppressing cy-
tokines, (iv) the locations of these cytokines within the CNS,
and (v) the temporal sequence in which a particular cell is
exposed to various cytokines (57). The actions of cytokines on
the vasculature in the brain also may be of pathophysiological
relevance. There is increasing evidence that IL-1 activates the
intercellular adhesion molecules, such as intercellular adhe-
sion molecule 1, vascular cell adhesion molecules, E-selectin,
and complement regulatory proteins, and it may also cause
platelet adhesion (34).
Previous studies of brain cytokine expression in cryptococ-
cosis have used mice infected via the natural route of infection
(the respiratory tract) or mice preimmunized with cryptococcal
antigen (soluble or heat-killed yeast) and subsequently chal-
lenged with C. neoformans via the intracerebral route. In both
situations, the immune response in the brain is a secondary
immune response (20, 23, 25). The intravenous inoculation of
naive mice with C. neoformans, as in the present study, results
in direct and preferential dissemination of the yeast to the
brain, allowing the primary immune response in the brain to be
followed. In most patients with cryptococcal CNS infection,
there is an inapparent preceding event which may be brief and
initiate a minimal immune response. The model of CNS infec-
tion after intravenous inoculation used in the present study
may approximate this situation. The model used in our studies
also provides a consistent level of fungal burden in the CNS
from mouse to mouse that is not afforded by the pulmonary
Using RT-PCR analysis, we found that mRNAs for cyto-
kines, such as TGF-?1 and IL-18, were expressed constitutively
and stayed at constant levels in infected and uninfected ani-
mals. This was also true of IL-12p35. A similar observation
about TGF-?1 has been made in patients with cerebral malaria
(9). This finding is consistent with the idea that TGF-?1, pro-
duced by endogenous glial cells, acts as an anti-inflammatory
and neuroprotective molecule and contributes to normal func-
tioning of the CNS (57). The expression of IL-1? at barely
detectable levels in some uninfected controls, but not in others,
is in accord with previous reports of whether this inflammatory
cytokine is constitutively produced in the brain (55). However,
the increase in expression during progressive infection ob-
served during our studies is similar to the pattern of expression
in the brain reported in studies of Toxoplasma gondii (27).
Normally, IL-12 is produced by monocytes and B cells (and
activated microglial cells in the CNS) and induces the produc-
tion of IFN-? by natural killer (NK) and T cells and the de-
velopment of a Th1 cellular immune response against infection
(15, 19). Neutralization of either IL-12 or IFN-? blocks the
development of protective CMI (23, 43). IL-12 has a positive
effect on host immune response, and the cytokine has been
shown to improve the treatment of cryptococcosis in a murine
model (13). Some investigators (16, 51) have detected IL-12p40
by immunohistochemistry and/or mRNA assay in normal neu-
ral tissue; other reports indicate that IL-12p40mRNA produc-
tion in the brain is not constitutive (48, 56). In two experiments
with three animals each, we showed constitutive expression of
mRNA for the IL-12p40subunit.
We also demonstrated the following. First, the transcripts
for the proinflammatory cytokines IL-1?, IL-1?, IL-6, and
TNF-?; the Th1 cytokine IFN-?; the Th2 cytokines IL-4 and
IL-10; and iNOS were induced above baseline levels, if any, in
the brain during the course of cryptococcal infection. Second,
the expression of IL-1?, TNF-?, and iNOS mRNAs showed
similar kinetics, with early detection on day 1 and increased
levels at later time points. Third, the expression of IL-4, IL-6,
and IFN-? mRNAs was detected on day 5, and expression of
IL-1? and IL-10 was detected on day 7. Fourth, the expression
of mRNA for IL-4, IL-10, and IFN-? remained constant in
later stages of infection. Similar results were obtained in the
murine model of cerebral candidiasis (3), in AIDS patients
with meningeal cryptococcosis, and in experimental murine
cryptococcal meningoencephalitis (36).
The initial expression of IL-12, IL-18, TGF-?1, IL-1?,
TNF-?, and iNOS transcripts prior to the appearance of ob-
servable infiltrating inflammatory cells suggests that these
mRNAs were produced by resident cells in the CNS. In con-
trast, IL-1?, IL-4, IL-6, IL-10, and IFN-? transcripts were
detected only after day 5 or 7 postinfection. Rises in the levels
of these transcripts corresponded with the initial arrival of
infiltrating cells (CD4?T cells, CD8?T cells, CD11b?mac-
rophages, and CD45R/B220?B cells) as observed by immu-
nohistochemistry, which requires significant numbers of cells
to be present before they can be detected in tissue sections.
However, the precise cellular origins of each cytokine remain
to be determined. Despite the infiltration of T cells, however,
IL-2 was not detected in the brains, suggesting that T-cell
proliferation may not occur.
TNF-? has effects similar to those of IL-1, and early pro-
duction of TNF-? is required to prevent the establishment of
cryptococcal foci in the CNS (23). Our data indicate that
TNF-? and iNOS are produced in direct response, and in
proportion, to the magnitude of the infectious burden seen in
the brain. A similar result was observed during the develop-
ment of cerebral candidiasis (3).
IFN-? is considered to play a critical role in the activation of
macrophages through induction of NO, a principal mediator
for macrophage killing activity in mice. In addition, IFN-?
enhances the antigen-presenting activity of macrophages and
acts as a stimulus for IL-12 production, causing the expansion
of a Th1 cell population. Protection observed in immunized
mice was mediated via IFN-? due to the activation of effector
cells already present at the site of infection or after their
recruitment to the site (1, 10). Treatment with IFN-? results in
a reduction in the cryptococcal burden in the CNS in infected
VOL. 72, 2004 CYTOKINE AND iNOS EXPRESSION IN MENINGOENCEPHALITIS 2347
mice (49), and based on these data, it has been possible to use
IFN-? in adjuvant immunotherapy (14, 37).
Three different forms of nitric oxide synthase that can affect
brain function have been identified in the brain: type I, neu-
ronal nitric oxide synthase; type II, iNOS; and type III, endo-
thelial nitric oxide synthase (33). iNOS is normally not ex-
pressed in the brain but is active for 4 to 8 h synthesizing NO
in nanomolar concentrations when induced (33, 50). NO acts
in part as a neurotransmitter and as a hormone but is cytotoxic
at high concentrations and might be involved in neural degen-
eration. In immune cells, NO is converted to products like
peroxynitrite, which are highly toxic to microorganisms (33).
IFN-?, TNF-?, IL-2, and IL-1? mediate the induction of ex-
pression of iNOS in microglia and astrocytes, whereas TGF-
?1, IL-4, and IL-10 inhibit iNOS induction (33). NO sup-
presses the functions of proinflammatory cytokines and
eliminates inflammatory cells in the CNS, possibly through an
apoptotic mechanism (34, 35, 57). This phenomenon has been
described in Paracoccidioides brasiliensis pulmonary infection;
in the early stage, NO is important for killing the organisms,
but persistent NO production likely contributes to the immu-
nosuppression observed during infection (8).
In view of our results with respect to the expression of IL-12,
TNF-?, IFN-?, and the infiltrating cells in the brain, we might
have expected a resolution of infection or at least a reduction
in the numbers of fungi in the brain. However, the concomitant
expression of TGF-?1, IL-4, and IL-10 might have acted as
immunosuppressive cytokines, allowing the continuation of the
infectious process. In addition, although in the early stages of
infection NO contributes to the killing of yeasts, the expression
of iNOS by endogenous cells may have been modulated by the
immunosuppressive cytokines or NO may cause immunosup-
pression itself, thereby permitting progression of the infection
and death. The former possibility is supported by the correla-
tion observed between levels of IL-4 and iNOS mRNAs. The
paradoxical depression of iNOS may be observed in the brain,
but not in the lungs, as the result of the neuroprotective action
of microglia, which express suppressive cytokines, such as
TGF-?1, to a greater degree than the proinflammatory cyto-
kines IL-1?, IL-6, IL-12, IFN-?, and TNF-? under natural
conditions (34, 35, 57).
C. neoformans virulence factors, such as the polysaccharide
capsule or melanin, can modulate phagocytosis, antigen pro-
cessing, cytokine production, lymphocyte proliferation, and
fungicidal activity (4, 38, 39, 45, 46, 52–54) and therefore
hinder the evolution of an effective immune response (2, 11).
Our finding of the absence of IL-2 production is consistent
with the impairment of local T-cell proliferation. In addition,
in the presence of specific antibody to the polysaccharide cap-
sule, microglial cells are potent effector cells against C. neo-
formans, resulting in prolonged survival and reduced organ
tissue fungal burden, as well as earlier and better-organized
granuloma formation in infected mice.
The fact that an avirulent strain, unlike a virulent strain,
induces the host to mount an effective Th1 response (5) that
subsequently protects against challenge with a virulent strain
demonstrates the importance of the pathogen in determining
the immune response. For example, mannoprotein from acap-
sular strains and nonmelanogenic C. neoformans strains pro-
motes Th1 responses that coincide with increased antifungal
activity of effector cells (5, 41, 52). Experimental studies with
different host genetic backgrounds showed that this also influ-
ences T helper cell differentiation (20, 47). CBA/J and CB-17
mice normally develop a Th1-type response to C. neoformans,
while C57BL/6 mice develop Th2-type responses during pul-
monary cryptococcal infection (19, 20, 25). A logical next step
will be to study the cytokine messages in the brains of different
mouse strains, infected with different Cryptococcus strains (dif-
fering in virulence and serotype), to characterize differences in
their immune responses in cerebral cryptococcal infection. The
activities of cytokines may not directly correlate with mRNA or
protein levels, as their function may be further modulated at
the level of posttranslational modification, processing multi-
mer assembly or release from the cell of origin, or presentation
or sequestration by specific carrier molecules and as a result of
varying receptor types on neighboring target cells. It will be
interesting to correlate the mRNA levels shown here with the
results of future studies measuring cytokine protein expression
to determine whether they correlate in the brain similarly to
the correlations described for expression in the lungs during
murine cryptococcosis (28).
In summary, we present new detailed information on the
timing of the mRNA expression of specific immune modula-
tory molecules in intracranial cryptococcal infection in the
murine experimental system. This should prove useful to other
investigators for studying the genetic bases of host resistance
or point to new interventions via therapy with cytokines (or by
elimination of cytokines with antibody or other inhibitors) to
promote more efficient elimination of the fungus from the
We thank Christopher A. Hunter and Roberto Macina for instruc-
tion and advice. We thank M. Martinez, M. Homola, P. Chu, L. Ellis,
and K. Gabriel for their assistance. We also thank Luiz de Souza for his
assistance with the statistical analyses.
Claudia M. L. Maffei was supported by a scholarship from Fundac ¸a ˜o
de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo (FAPESP).
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