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Departments of
*
Pathology and
Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131;
Division of Pulmonary Medicine, University of Michigan Medical School, Ann Arbor, MI; and
§
Torrey Pines Institute for Molecular Studies, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and NO. Clearance in congenic BALB/c mice proceeds more slowly
than in C.B-17 mice, even though the only genetic difference between
these strains is at the Ig H chain-containing region of chromosome 12.
Examination of the pulmonary immune response in the two strains
revealed that both cleared lung Cne by T cell-dependent mechanisms and
generated equivalent levels of NO. Furthermore, both strains recruited
equal numbers of macrophages, lymphocytes, and neutrophils to the
lungs, although BALB/c mice recruited higher numbers of eosinophils.
Notably, leukocytes isolated from BALB/c lungs during infection
secreted lower levels of IFN- and higher levels of the Th2 cytokines
IL-4 and IL-5 as compared with lung leukocytes from C.B-17 mice.
Furthermore, serum levels of IgM, IgG1, IgG2a, and IgG3 anti-Cne
Abs generated during infection were significantly greater in BALB/c
mice than C.B-17 mice. These data suggest that although both BALB/c and
C.B-17 mice clear pulmonary cryptococcosis through T cell-mediated
mechanisms, Ig H chain-linked genes in BALB/c mice are associated with
a decreased effectiveness of the host response, which we suggest might
influence the balance in Th1/Th2 T cell subset development or increase
anti-Cne Abs, or both. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have established a murine pulmonary Cne infection model in which the
organism is inoculated into the trachea to dissect mechanisms involved
in a protective pulmonary immune response (3).
Pulmonary clearance of a moderately virulent Cne strain, 52D,
varies among immunologically intact mouse strains, providing an
opportunity to identify genes that play a role in regulating pulmonary
immunity against this fungal pathogen. C.B-17 mice clear 52D, and
clearance depends on the development of a Th1-type response to Cne and
production of IFN- (4, 5, 6). IFN- is required for the
activation of recruited macrophages to produce NO. Thus, if
anti-IFN- Ab is administered after the initial development of an
immune response and before full activation of recruited macrophages, or
if the NO pathway is inhibited by treatment with the arginine analogue,
NG-monomethyl-L-arginine, lung
clearance is abrogated (6).
BALB/c mice also clear pulmonary cryptococcal infections with Cne 52D (3). However, preliminary studies indicated that at early time points in the infection, BALB/c mice consistently had more Cne in their lungs than C.B-17 mice, although both mouse strains reduced their lung burdens to similar low numbers late in the infection. The two mouse strains are genetically identical except at the telomeric region of chromosome 12, which, in addition to other genes, encodes the Ig H chain locus (IgH) (7).
The goal of the current study was to determine whether the genetic differences encoded in the unshared regions of chromosome 12 in BALB/c and C.B-17 mice influenced the T cell immune response generated against Cne or, alternatively, whether Ab levels might differ and perhaps suggest a role for Ab in lung clearance. The results demonstrated that although both strains required T cells to initiate cryptococcal lung clearance and produced similar amounts of NO at the same rate, they differed in lymphocyte cytokine secretion profiles and in serum anti-Cne Ab levels during infection. Therefore, an IgH-linked gene (or genes) on chromosome 12 influenced the rate of lung clearance, T cell subset development, and serum anti-Cne Ab levels during a pulmonary cryptococcal infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HyClone RPMI 1640 media, HBSS, and Dulbecco’s PBS (HyClone, Logan, UT) were used throughout these studies. Media was certified as endotoxin free by vendor, supplemented with 100 U/ml penicillin/ streptomycin and 2 mM L-glutamine (Life Technologies, Grand Island, NY), 1 mM sodium pyruvate and 1 mM nonessential amino acids (Cellgro-Fisher Scientific, Pittsburgh, PA), and 5–10% FBS (Life Technologies) as noted below. FBS was heat-inactivated at 56°C for use in assays. Anti-CD4 (GK1.5) and anti-CD8 (YTS 169.4) mAbs, were produced as ascites in SCID mice and purified on a protein A-Sepharose affinity chromatography column (BioRad Laboratories, Rockville Center, NY) as previously described (8).
Mice
C.B-17 and BALB/c mice were raised in the University of New Mexico Animal Resource Facility. Some BALB/c mice were also obtained from The Jackson Laboratory (Bar Harbor, ME.) or Harlan Laboratories (Indianapolis, IN). Mice, 6–8 wk old, were housed in filter top cages in a pathogen-free environmental unit. Sterile food and water were given ad libitum. Sentinel mice from breeding colonies and experimental areas are routinely analyzed serologically and histologically for evidence of secondary infections. For urinary nitrate studies, mice were housed (two mice/cage) in Nalgene metabolic cages (Ancare, Bellmore, NY) and placed on a defined nitrate-free amino acid diet (Zeigler Brothers, Gardners, PA).
Organism
The moderately encapsulated, serotype D Cne strain, 52D (no. 24067; American Type Culture Collection, Manassas, VA), was maintained as previously described on slants of Sabouraud dextrose agar (1% neopeptone, 4% dextrose, 1% agar; Difco, Detroit, MI). For use in assays, Cne was cultured in Sabouraud dextrose broth (1% neopeptone, 2% dextrose) on a shaker for 36–40 h at 25°C. In preparation for inoculation, yeasts were washed three times with pyrogen-free saline (McGaw, Irvine, CA) and counted on a hemocytometer.
Cryptococcal infection model
Mice were inoculated intratracheally (i.t.) as previously described (3). Briefly, mice were anesthetized, a small incision was made above the trachea, and 2–3 x 103 yeasts in 50 µl of pyrogen-free saline were deposited into the trachea using a bent 30-gauge needle. For urinary nitrate studies, urine was collected daily from mice housed in metabolic cages. The volume was measured and stored at -70°C. For T cell depletion studies, mice were given a total of 300 µg of anti-CD4 and 300 µg of anti-CD8 mAbs as three i.p. injections of 100 µg of each mAb on days -1, 0, and +1 of infection. Control mice were treated with an equivalent amount of rat Ig (predominantly IgG; The Jackson Laboratory). Depletion was maintained by repeated i.p. injections of 100 µg of each mAb every 7 days and monitored by flow cytometry of splenic cells as previously described (8).
All protocols were approved by the Internal Review Board for Animal Rights Committee. In this Cne model, animals do not demonstrate signs of infection until after 28 days, and then only in some strains of mice and in immunocompromised mice. Experiments were terminated before evidence of severe animal distress.
Quantitation of Cne in lung
At various time points after i.t. inoculation, lungs were removed and homogenized in sterile water. Serial dilutions were plated on Sabouraud dextrose agar plates. Plates were incubated for 48–72 h at 25°C, and the number of CFU were determined. The number of Cne initially deposited in the lung was determined by culturing CFU from lungs obtained 15 min after inoculation.
Isolation of lung and lung associated-lymph node (LALN) cells
Lung cells were isolated from individual infected mice. The mice were pretreated with heparin i.p. (150 U; Elkins-Sinn, Cherry Hill, NJ) 10 min before being euthanized. The pulmonary vasculature was perfused with sterile saline to eliminate peripheral blood cells, and the lungs were removed, minced, and incubated with collagenase (0.7 mg/ml in RPMI with 5% FBS; Boehringer Mannheim Biochemicals, Indianapolis, IN) and DNase (30 µg/ml type IV bovine pancreatic DNase I; Sigma, St. Louis, MO) for 90 min at 37°C. Digested lungs were tapped through a wire mesh. Large particulate matter was removed by passing the cell suspension through a loose nylon wool plug. Cells were washed twice with HBSS and resuspended in RPMI with 5% FBS. Red cells were lysed, if necessary, using an ice-cold isotonic 0.14 M ammonium chloride solution (pH 7.4). Cells were counted on a hemocytometer and cell types were determined by performing differentials on Wright-Giemsa-stained cytospin preparations (300 cells/slide were counted). For cytokine secretion studies, lung cells were spun through a 30% Percoll/DPBS solution (Pharmacia, Piscataway, NJ) to eliminate red cell ghosts and cellular debris from the lung cell preparation. Cells were incubated on 100 mm2 tissue culture plates for 2 h in 5% FBS/complete RPMI 1640 at 37°C in 5% CO2 to remove adherent cells. Nonadherent cells were collected, pelleted, resuspended in RPMI 1640 with 10% FBS, and counted on a hemocytometer with trypan blue to enumerate live cells. To prepare LALN cells for cytokine analysis, LALNs were removed from each mouse and disrupted between two sterile, frosted glass slides to obtain a single-cell suspension. The LALN cells were washed with HBSS and resuspended in RPMI 1640 with 10% FBS.
Cytokine secretion cultures
Lung leukocytes were prepared as described above and cultured in RPMI 1640 with 10% FBS supplemented with 2 µg/ml amphotericin B (Sigma) to block Cne growth in culture, 2.5 x 10-5 M 2-ME (Eastman Kodak, Rochester, NY), 1 µg/ml indomethacin (Sigma), and 250 U/ml catalase (Worthington Biochem, Freehoff, NJ). Amphotericin B was previously found to have no effect on the secretion of cytokines in 24 h cultures, but any effect in the 48 h cultures should be uniform for both C.B-17 and BALB/c cell cultures. Indomethacin and catalase are consistently used in lung cell cultures to avoid the effects of PG and oxygen radicals on suppression of lymphocytes in cultures. Again, the uniform use of these reagents in cultures of cells from both strains compared should allow for valid comparisons. Cells were cultured in duplicate at 5 x 106 cells/ml in 24-well tissue culture plates and either cultured in medium alone or stimulated with 5 µg/ml Con A (Sigma). In some experiments, 1 µg/ml of anti-IL-4 receptor Ab (Genzyme, Cambridge, MA) was added to cultures to optimize assessment of secreted IL-4. LALN cells were cultured as above except at 5 x 106 cells/ml in duplicate in 96-well plates and cultured in either media alone or with added Con A. Supernatants were collected at 48 h for cytokine analysis.
Cytokine ELISAs
Cytokines were analyzed using a two-site sandwich ELISA. Capture
mAbs for IL-4 (11B11), IL-5 (TRFK5), and IFN- (R46A2) were obtained
from PharMingen (San Diego, CA) and bound to ELISA plates diluted in
0.1 M Na2HPO4 solution (pH
9.0). Nonspecific binding was blocked with a 1% BSA/PBS solution.
Biotinylated detection mAbs included: anti-IL-4 (BVD6-24G2),
anti-IL-5 (TRFK4), and anti-IFN- (XMG1.2; PharMingen).
Streptavidin-HRP (1 mg/ml) in blocking buffer was added to detect bound
cytokines in the assays and developed using an ABTS
(azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) solution, and the OD
at 405 nm was determined. Cytokines were quantified by comparison to
standard curves using recombinant IL-4, IL-5, and IFN- (PharMingen).
An internal standard was included to monitor reproducibility of ELISAs
using recombinant IL-4, IL-5, and IFN- obtained from Genzyme.
Detection limits for each cytokine were assigned as the lowest
concentration in the linear portion of the standard curve: IL-4 (0.25
ng/ml), IL-5 (1 ng/ml), and IFN- (1.56 ng/ml).
Nitrite/nitrate assays
Urine nitrate was reduced to nitrite using nitrate reductase of Pseudomonas oleovorans (no. 8062; American Type Culture Collection) as previously described (9), and nitrite levels were then determined using the Griess assay (10).
Detection of anti-Cne-reactive Ab
A loopful of Cne strain 52D was removed from a Sabouraud dextrose agar slant (in stationary phase) and washed three times with saline. Then, 1 x 106 Cne were incubated with a 1/10 dilution of mouse sera from mice obtained from Harlan or our colony for 30 min on ice in a total volume of 50 µl staining buffer (1% FCS-PBS). Cne were washed three times with staining buffer before the addition of 50 ml FITC-labeled goat F(ab')2 anti-mouse IgG1, IgG2a, or IgG3 (Southern Biotechnology Associates, Birmingham, AL) at a final concentration of 10 µg/ml, or FITC-goat F(ab')2 anti-mouse IgM (Organon Teknika, Durham, NC) at a final dilution of 1/100. All secondary Abs were H chain specific. Cells were incubated on ice for 30 min, washed three times with staining buffer, and fixed with 0.5% paraformaldehyde. Cell-associated fluorescence was measured with a Becton Dickinson FACScan, and data was analyzed using PC-LYSIS software. To establish a standard curve, mAbs specific for Cne capsular polysaccharide and representing each of the four isotypes to be quantified were incubated with Cne at 1.0, 0.1, and 0.01 µg/ml and detected with the appropriate FITC-labeled secondary. Mean fluorescence intensity was plotted against concentration, and a curve was generated. The mean fluorescence intensity of each mouse serum was interpolated using the standard curve to determine the concentration. IgG1 anti-Cne (439) was a gift from Dr. Thomas Kozel. IgG2a (2D10-Ig2a), IgG3 (3E5), and IgM (11E2) anti-Cne Abs were gifts of Dr. Arturo Casadevall.
Statistical analysis
Differences in all measured variables between BALB/c and C.B-17 mice were analyzed using unpaired t tests employing Statview software (Abacus Concepts, Berkeley, CA) with the exception of differences in the levels of anti-cryptococcal Abs. Differences in Ab levels between strains were analyzed by ANOVA after rank transformation of the data (Excel; Microsoft, Redmond, WA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies demonstrated that after i.t. deposition, Cne
proliferates in the lungs of resistant C.B-17 mice during the first
week of infection. A decrease in lung CFU begins between day 7 and 14,
and by day 90, organisms are often below the level of detection (<50
CFU) (6, 11), suggesting that these mice may eventually
completely clear the infection. Comparison of Cne clearance in BALB/c
and C.B-17 mice revealed that from day 14 until day 56, BALB/c mice had
significantly higher numbers of Cne as compared with C.B-17 mice (Fig. 1
A). Thus, although at day
84 both mouse strains reached very low and similar CFU, pulmonary
clearance progressed less efficiently in BALB/c mice.
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To determine the relative contribution of T cells in Cne pulmonary
clearance in BALB/c as compared with C.B-17 mice, mice were treated
with a combination of anti-CD4 and anti-CD8 mAbs before
infection with Cne. T cell depletion was maintained by weekly
injections of mAbs and monitored by flow cytometric analysis of
splenocytes. Removal of T cells in BALB/c or C.B-17 mice abolished
clearance of Cne during the first 21 days of infection (Fig. 1
B). The Cne lung burden in T cell-depleted mice of both
strains was significantly different from control Ig-treated mice by day
21 and were similar to previously reported levels observed in SCID mice
(3, 6). These results indicated that clearance of Cne in
both BALB/c and C.B-17 mice was T cell mediated.
Cytokine production by LALN cells and lung leukocytes in BALB/c and C.B-17 mice
Although T cells were necessary for clearance, the less efficient
Cne clearance in BALB/c mice might reflect a difference in T cell
cytokines produced during infection between the two mouse strains. This
would be important, because IFN- was shown to be required both in
the early inductive phase of the immune response (5) and
during the effector stage (6). The levels of IFN-,
IL-4, and IL-5 were determined for both BALB/c and C.B-17 mice by
analyzing the cytokines produced by Con A-stimulated LALN cells and
lung leukocytes isolated from infected mice both before (7 days) and at
the time (14 days) clearance begins. LALNs in uninfected mice were too
small to be reliably studied. Con A-stimulated lung leukocytes isolated
from uninfected C.B-17 or BALB/c mice secreted low to undetectable
cytokine levels, and no differences were detected between strains.
Analysis of the cytokines produced by LALN cells isolated from infected
BALB/c and C.B-17 mice revealed that BALB/c LALN cells secreted
significantly lower levels of IFN- at day 7 and 14 of infection as
compared with LALN cells from C.B-17 mice (Fig. 2). LALN cell secretion of IL-4 and IL-5
was not detected in either BALB/c or C.B-17 mice. Cytokines were not
detected in cultures of unstimulated LALN cells. Quantitation of
cytokines produced by leukocytes isolated from the lung during
infection revealed lower levels of IFN- produced by BALB/c lung
leukocytes at both days 7 and 14 of infection as compared with C.B-17
lung leukocytes (Fig. 3A).
Similar levels of IL-4 were produced by lung leukocytes from both mouse
strains at day 7, but by day 14, production of IL-4 by BALB/c lung
leukocytes was significantly greater than by C.B-17 lung leukocytes
(Fig. 3B). Higher levels of IL-5 were also produced by
BALB/c lung leukocytes by day 14 as by C.B-17 lung leukocytes (Fig. 3C).
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An important role for T cells in the immune response against Cne
is to facilitate maximal recruitment of effector cells to the lung
(6, 12). To determine whether the difference in cytokine
production between BALB/c and C.B-17 mice affected cellular
recruitment, the numbers and types of leukocytes present in the lung
during infection were examined. Quantification of lung cells isolated
from BALB/c and C.B-17 mice verified that similar numbers of cells were
recruited to the lungs of both mouse strains (Fig. 4A) and that similar numbers
of lymphocytes, macrophages, and neutrophils were present at the peak
of leukocyte recruitment on day 14 (Fig. 4B). However,
significantly greater numbers of eosinophils were present in BALB/c
lungs than in C.B-17 lungs at day 14 (p <
.0001).
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Although there was significantly less IFN- produced by
leukocytes from BALB/c mice, this cytokine was detected in both mouse
strains. One important function of IFN- in protection against Cne is
to activate macrophages to express the enzyme, inducible NO synthase,
and secrete NO (6). NO production was determined by
monitoring urinary nitrate excretion in both Cne-infected BALB/c and
C.B-17 mice in parallel. The levels of excreted urinary nitrate
observed in BALB/c and C.B-17 mice were similar at all time points
(Fig. 5). The levels of inducible NO
synthase protein were shown to increase in the lungs of both mouse
strains between day 0 and day 14 of infection by Western analysis (data
not shown). In addition, isolated lung cells were analyzed by
immunohistochemistry for inducible NO synthase, and a similar
percentage of positive-staining macrophages were found in both
14-day-infected BALB/c and C.B-17 mice (BALB/c, 36.8 ± 19.4%;
C.B-17, 26.6 ± 13.0%; p > 0.1,
n = 5 mice/strain).
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The finding that systemic NO levels were similar between BALB/c
and C.B-17 mice suggested that the difference in Cne clearance could
not be completely explained by IFN- secretion differences, because
our previous studies had shown the dependence of clearance on
IFN--dependent NO production (6). Another possible
explanation was that Ab influenced Cne clearance. Sera from uninfected
and Cne-infected mice were incubated with Cne strain 52D, and the
presence of bound IgM and IgG1, IgG2a, and IgG3 isotypes was detected
by indirect immunofluorescence. Surprisingly, sera from all uninfected
BALB/c mice possessed IgM, and many also had IgG3 Cne-reactive
Ab-depending on the age of the mice (Fig. 6, A and B). In
contrast, sera from uninfected C.B-17 mice exhibited little Cne-binding
activity. During infection, the levels of IgM Abs in BALB/c mice
remained stable for the first week of infection and then rose 3- to
4-fold during the second week before declining, but were not detectable
in C.B-17 mice (Fig. 6A). Although some increase in IgG
anti-Cne Ab levels was observed in C.B-17 serum during infection,
BALB/c mice had higher levels of IgG anti-Cne Abs of all three
isotypes (Fig. 6, B–D). The predominant IgG isotype in both
mouse strains was IgG3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and subsequent NO production (4, 5, 6). We found in the
current study that clearance of Cne in both BALB/c and C.B-17 mice was
dependent on the presence of T cells, and both strains developed a
Th1-type immune response as shown by production of IFN- by LALN
cells and lung leukocytes, recruitment of similar numbers of
lymphocytes and macrophages into the lung during infection, and
activation of recruited macrophages to produce NO. However, BALB/c mice
also demonstrated characteristics of an accompanying Th2 response to
Cne with increased lung leukocyte secretion of IL-4 and IL-5, a higher
number of eosinophils in their lungs, and higher anti-Cne Ab levels
(including increased IgG1 anti-Cne Abs). Another difference between
the two mouse strains was the presence of Cne-reactive Abs in
uninfected BALB/c mice, but not in uninfected C.B-17 mice. The results
indicate that a gene(s) on chromosome 12 controls expansion of
Cne-reactive B cells and Ab production in uninfected mice and
influences humoral and cellular immune responses during a Cne pulmonary
infection and fungal lung clearance. The region of chromosome 12 that differs between BALB/c and C.B-17 mice encompasses many genes including a minor histocompatibility Ag (H40) (13), some alloantigens (TSU, Lm-1) (14, 15), the bacillus Calmette-Guérin-anergy gene (16), the IgH complex (7), and others with unknown functions. Although the bacillus Calmette-Guérin-anergy gene can negatively affect immunity, this gene is unlikely responsible for the reduced ability of BALB/c mice to clear Cne, because anergy occurs in mice with the IgHb haplotype and BALB/c mice have the IgHa haplotype (16). Preliminary studies using C.B-reduced congenic strains with smaller chromosome 12 region disparities than exist between C.B-17 and BALB/c mice suggests that the Cne clearance differences between these two strains map to the IgH locus (J.A.L., unpublished observations). Molecular characterization of several mAbs against cryptococcal polysaccharide (CNPS) has revealed a relatively limited Ab repertoire with restricted Ig variable gene element usage for both H and L chains (17, 18, 19). The apparent oligoclonal Ab response to CNPS suggests that very few dominant, immunogenic epitopes of CNPS are recognized. Thus, genetic differences at the IgH locus could affect the potential of mouse strains to generate Ab capable of recognizing Cne and explain the finding of higher anti-CNPS Ab levels in BALB/c mice as compared with C.B-17 mice. We believe that uninfected BALB/c mice exhibit CNPS-reactive IgM and IgG3 Ab, because they are exposed to a foreign, cross-reactive polysaccharide in their environment; and that CNPS, as a T-independent type-2 Ag (20, 21), directly restimulates these cross-reactive B cells upon exposure to the organism during infection. It is also possible that the Ab and reactive B cells are the result of exposure to low levels of the ubiquitous cryptococcal yeast in their bedding, but in any case, both mouse strains are housed in the same animal care facilities, and only the BALB/c mice are able to respond in this fashion.
Our earlier studies suggested that the development of a Th1 response against Cne was not linked to the IgH-linked region of chromosome 12, because C57BL/6 mice, which are incapable of either clearing Cne or developing a Th1 immune response to pulmonary Cne (4), are identical with C.B-17 mice at this region (16). The data in the current paper, coupled with our previous studies, indicate that genes outside of the chromosome 12 region are the most important in defining whether cryptococci will be cleared from the lung or not. If the resistance phenotype is present as in C.B-17 and BALB/c mice, then the presence of IgH-linked genes from either the BALB/c or the C57BL/6 mouse should define the rate of pulmonary clearance. Therefore, the C.B-17 mouse represents a strain that has the full complement of resistance genes of the BALB/c mouse with the replacement of its IgH-linked genes by C57BL/6 chromosome 12, and a more favorable clearance pattern occurs than in the wild-type BALB/c.
The ability of BALB/c mice to make Cne-cross-reactive B cells and Ab before and early in infection in BALB/c mice may explain the difference in T cell subset development between BALB/c and C.B-17 mice. The differentiation of naive, Ag-specific CD4+ Th cells into mature Th1- or Th2-type cells depends on several factors including the type of APC, the cytokines present in the milieu at the time of Ag presentation, and the Ag dose. Although dendritic cells are the major APCs involved in initiating a primary immune response (22), primed Ag-specific B cells can also function as APC for naive T cells (23, 24, 25). Dendritic cells secrete IL-12 and tend to induce development of Th1 cells (26), while Ag presentation by B cells favors development of Th2 cells (24). Although dendritic cells likely constitute the majority of cells presenting Cne Ag to naive T cells in LALNs and induce the development of Th1 cells, we cautiously propose that Ag presentation by an expanded population of Cne-reactive B cells in BALB/c mice could alter the ratio of Cne-specific Th1:Th2 cells and explain the higher levels of Th2 cytokines produced by LALN and lung leukocytes from BALB/c mice. A second possibility to explain how the presence of Ig could affect T cell development is that cross-reacting Ab results in sequestration of cryptococcal Ag, thereby reducing the concentration of Ag in lymph nodes during the developing response in BALB/c mice as compared with C.B-17 mice and favoring Th2 subset development (27).
We have offered postulates for how the chromosome 12 locus could
influence Ig levels and T cell cytokine profiles, but the mechanism
responsible for the less efficient clearance of Cne in BALB/c mice
remains uncertain. However, at least three possibilities exist. First,
although NO production is necessary, it may not be sufficient for Cne
clearance, and the lower levels of IFN- produced by BALB/c
leukocytes may reflect a decrease in additional IFN--dependent
mechanisms that synergize with NO for efficient reduction of Cne.
Second, the generation of Th2 cytokines may adversely affect the
ability of BALB/c mice to clear Cne by suppressing the aforementioned
IFN--dependent clearance mechanisms or activating effector cells,
such as eosinophils, to inhibit clearance. A third possibility is that
the anti-Cne Abs generated in BALB/c mice have a direct inhibitory
effect. Studies by others examining individual anti-CNPS mAb have
shown that certain mAb prolonged survival and others decreased survival
when passively administered before Cne inoculation in lethal i.v.,
i.p., or pulmonary Cne infection models (19, 28, 29, 30, 31). In
these studies, the capacity of a particular mAb to be protective
depended on both epitope specificity and isotype (28, 29, 30, 32). Interestingly, particular IgG3 anti-CNPS mAbs were not
protective and could even interfere with the efficacy of a protective
Ab (33), perhaps by sequestering Cne in unactivated
macrophages via Fc receptor-dependent phagocytosis (32, 34). Further studies are underway to analyze the immune response
to Cne in C.B-reduced congenic strains to identify the specific gene(s)
that determine the difference between the two strains. In addition, we
will examine IgH knockout mice on the BALB/c background to determine
whether these mice demonstrate cryptococcal lung clearance curves
identical with C.B-17 mice.
Overall, these studies reveal that an IgH-linked gene(s) on chromosome 12 can influence humoral and cellular immune responses to Cne and subsequently affect the ability of fully immunocompetent mice to respond to this opportunistic organism. By extension, the increased susceptibility to Cne infections of some apparently immunocompetent humans may reflect subtle, genetically determined differences in their immune response to this pathogenic yeast.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mary F. Lipscomb, Chairperson, Department of Pathology, University of New Mexico, School of Medicine, 915 Camino De Salud, NE, Albuquerque, NM 87131-5301. E-mail address:
3 Abbreviations used in this paper: Cne, Cryptococcus neoformans; IgH, Ig H chain; i.t., intratracheally; LALN, lung-associated lymph node; CNPS, cryptococcal polysaccaride
Received for publication April 9, 1999. Accepted for publication July 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are required for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C.B-17 mice. Am. J. Respir. Cell Mol. Biol. 17:733. interferon-induced nitric oxide in pulmonary clearance of Cryptococcus neoformans. Am. J. Respir. Cell Mol. Biol. 13:116.[Abstract]
ß transgenic model. J. Exp. Med. 182:1570.
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D. O. Beenhouwer, S. Shapiro, M. Feldmesser, A. Casadevall, and M. D. Scharff Both Th1 and Th2 Cytokines Affect the Ability of Monoclonal Antibodies To Protect Mice against Cryptococcus neoformans Infect. Immun., October 1, 2001; 69(10): 6445 - 6455. [Abstract] [Full Text] [PDF]
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 J. A. Wilder, D. S. Collie, D. E. Bice, Y. Tesfaigzi, C. R. Lyons, and M. F. Lipscomb Ovalbumin aerosols induce airway hyperreactivity in naive DO11.10 T cell receptor transgenic mice without pulmonary eosinophilia or OVA-specific antibody J. Leukoc. Biol., April 1, 2001; 69(4): 538 - 547. [Abstract] [Full Text]
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S. Neuville, O. Lortholary, and F. Dromer Do Kinetics of the Humoral Response to Cryptococcus neoformans Proteins during Murine Cryptococcosis Reflect Outcome? Infect. Immun., June 1, 2000; 68(6): 3724 - 3726. [Abstract] [Full Text] [PDF]
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14 mice/strain/time point). *, Significant
difference in lung CFU between BALB/c and C.B-17 mice
(p < 0.05). B, Cne clearance in T
cell-depleted and control BALB/c and C.B-17 mice. Mice were depleted of
T cells by treatment with a combination of anti-CD4 and CD8 mAbs
before infection and every 7 days thereafter. Control mice were treated
with an equivalent amount of rat Ig. The data represents the average
mean lung CFU ± SEM from three experiments (n
= 7–13 mice/strain/time point). **, Significant difference in lung
CFU between control and T cell-depleted (T-) BALB/c and C.B-17 mice
(p < .0001).
0.005).
