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,
*
Department of Microbiology and Immunology and
University of Massachusetts Medical School, Worcester, MA 01605; and Departments of
Pathology and
Medicine, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, monocyte chemoattractant protein-1, and
macrophage-inflammatory protein-1
than parental mice. Ab
administration had different effects on infected NOS2-/-
and parental mice with respect to IFN-, monocoyte chemoattractant
protein-1, and macrophage-inflammatory protein-1 levels. Ab
administration increased lung levels of IFN- in parental mice and
reduced levels in NOS2-/- mice. The results indicate that
NO is involved in the regulation of cytokine expression in response to
cryptococcal pneumonia and is necessary for Ab efficacy against
C. neoformans in mice. Our findings indicate a complex
relationship between Ab efficacy against C. neoformans
and cytokine expression, underscoring the interdependency of cellular
and humoral defense mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For C. neoformans, several potential mechanisms of Ab-mediated protection have been described, including enhancement of phagocytosis by macrophages (8), complement activation (9), and clearance of polysaccharide Ag, which causes a variety of deleterious effects on the host immune response (10, 11, 12). Mice infected with C. neoformans and given specific Ab mount a more intense pulmonary granulomatous response than control mice, suggesting that Ab-mediated protection involves enhancement of cellular immunity (13). Hence, the current consensus is that humoral immunity protects against C. neoformans by enhancing the efficacy of the cellular immune response (14). In support of this, in vitro studies have shown that Ab binding to the capsule can enhance Ag presentation and induce changes in cytokine and costimulatory molecule expression (15, 16). However, the mechanism by which Ab alters the inflammatory response to C. neoformans in vivo and the variables that affect the outcome of humoral-cellular collaboration are unknown.
The generation of NO by inducible NO synthase (NOS2)3 has been implicated in the antimicrobial activity of activated macrophages against a variety of intracellular pathogens, including M. tuberculosis (17), Leishmania major (18), and L. moncytogenes (19). Macrophages play a central role in host defense against C. neoformans through both oxygen- and nitrogen-derived molecular mechanisms (20, 21). Depending on the concentration, chemically generated NO is fungistatic or fungicidal for C. neoformans (22). Furthermore, NO derived from cultured human astrocytes is fungistatic against C. neoformans (23), and the appearance of NOS expression in tissue is associated with the clearance of C. neoformans in immunocompetent mice and rats (24, 25). Hence, there is considerable evidence that NO is important for host defense against C. neoformans.
Because NO is important for host defense against C. neoformans and because Ab appears to depend on an effective cellular response for activity, we examined the efficacy of Ab administration against cryptococcal infection in mice with a targeted deletion of NOS2. NOS2-/- mice were found to be more susceptible to C. neoformans infection, and Ab administration was ineffective in prolonging survival. Our results provide additional support for the importance of NO in defense against C. neoformans and demonstrate, for the first time, a dependence of Ab function on NO.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strain ATCC 24067 (serotype D; American Type Culture Collection (ATCC), Manassas, VA) was grown from frozen stock in Sabouraud’s dextrose broth (Difco, Detroit, MI) at 30°C for 48 h. This strain was selected because it has been extensively used in Ab studies and is well characterized (26). Inoculum was confirmed by counting CFUs on Sabouraud dextrose agar (Difco). Glucuronoxylomannan (GXM) was isolated from culture supernatant of strain 24067, as described (27).
Antibodies
mAb 2H1 (IgG1) binds the capsule polysaccharide GXM of C. neoformans (28). This mAb protects mice against C. neoformans infection in a variety of infection models (13, 28, 29, 30). Murine ascites containing mAb 2H1 protein was prepared by injecting 2H1 hybridoma cells into the peritoneum of pristine-primed BALB/c mice. NSO (control) is the nonproducing mouse myeloma fusion partner of the hybridoma 2H1, and ascites produced using this cell line was used in some experiments. For some experiments, mAb 2H1 was purified from ascites fluid using protein G affinity chromatography (Pierce, Rockford, IL), as instructed by the manufacturer, and PBS was used as the control.
Mice
Breeding pairs of C57BL/6J x 129/SvEv (parental) and NOS2-/- mice (gift of C. Nathan, Weill Medical College of Cornell University, New York, NY) were maintained at the Animal Institute of Albert Einstein College of Medicine. Additional 6- to 8-wk-old female C57BL/6J x 129/SvEv (parental-F1), C57BL/6J, and NOS2-/- were obtained from The Jackson Laboratory (Bar Harbor, ME). These mice were kept in a specific pathogen-free barrier facility in microisolator cages, fed irradiated rodent food, provided with autoclaved bedding, and routinely monitored for serologic evidence of exposure to common murine pathogens. All serological testing was negative.
Macrophage assays
Alveolar and peritoneal macrophages were isolated from parental and NOS2-/- mice. For alveolar macrophage isolation, the tracheas of euthanized mice were exposed by a skin incision, and a 20-gauge angiocath (BD Biosciences, Sandy, UT) was advanced into the trachea 3 mm and sutured in place. The lungs were then lavaged 10 times through the catheter with sterile calcium and magnesium-free HBSS without Phenol Red (Life Technologies, Grand Island, NY) with 1 mM EGTA (Sigma-Aldrich, St. Louis, MO) using 0.8 ml per wash (31). The lavage fluids were pooled, and cells were collected by centrifugation. Peritoneal macrophages were collected from the same mice by washing the abdominal cavity five times with HBSS using a Pasteur pipette. The total cell suspension was collected by centrifugation, and erythrocytes were lysed by resuspending in ice-cold 0.17 M NH4Cl and incubating on ice for 10 min. A 10-fold excess of RPMI 1640 solution was then added to make the solution isotonic, the cells were collected by centrifugation and suspended in staining solution (PBS, 1% FBS), and live cells (trypan blue exclusion) were counted in a hemocytometer chamber. The cells were then suspended in DMEM (Life Technologies), 10% NCTC-109 medium (Life Technologies), and 1% nonessential amino acids (Cellgro; Mediatech, Washington, D.C.) containing penicillin and streptomycin (Life Technologies). The cells were plated at a density of 8 x 104/well in a 96-well tissue culture plates, and incubated overnight at 37°C.
The protocols for macrophage phagocytosis and killing assays have been
described (13). Briefly, macrophages were stimulated
overnight with 100 U/ml murine rIFN- (Boehringer Mannheim,
Indianapolis, IN). The media were replaced with fresh media that
contained 100 U/ml IFN- and 1 µg/ml LPS (Sigma-Aldrich).
Phagocytosis was measured in media with or without 10 µg/ml mAb 2H1.
C. neoformans cells were added at a macrophage to yeast
ratio of 5:1, and the suspensions were incubated at 37°C for 2 or
4 h. The macrophage monolayer was then washed several times with
sterile PBS, fixed with cold absolute methanol, and stained with 1:20
solution of Giemsa. The phagocytic index was determined by microscopic
examination at a magnification of x600. The phagocytic index is the
number of attached and ingested cryptococci divided by the number of
macrophages per field. Four fields were counted in four wells for each
measurement. For killing assays, activated macrophages and C.
neoformans were coincubated for 18 or 24 h at 37°C. Cell
supernatants were collected and cells were lysed by addition of 0.1 of
sterile distilled water to each well and incubating for 30 min at room
temperature, followed by aspirating and ejecting the lysate with a
pipette several times to complete cell disruption. PBS (0.1 ml) was
used to rinse each well, and the cell supernatant, lysate, and rinse
from each well were pooled, vortexed, diluted, vortexed again, and
spread on Sabouraud’s dextrose agar (32). Nitrite in cell
supernatant was measured using Griess reagent (1% sulfanilamide, 0.1%
N-1-napthylethylenediamine, 2.5% phosphoric acid) after
reducing nitrate to nitrite with Pseudomonas
oleovorans nitrate reductase, as described
(33).
In vivo studies
Survival analysis. Parental and NOS2-/- (6- to 8-wk-old) mice were infected intratracheally (IT) with 106 C. neoformans, as previously described (13). Ab was administered i.p. 24 h before infection. Mice were monitored daily for mortality and morbidity. On day 107, surviving mice were killed, their lungs were removed, and the right upper lobes were fixed in 10% buffered formalin (Fisher, Pittsburgh, PA) for histology.
CFU experiments. Parental and NOS2-/- mice were given mAb and infected, as described above. At day 14 postinfection, mice were killed by cervical dislocation and organ CFUs were determined by homogenizing the tissue and plating on Sabouraud’s agar, as described (13). Blood was obtained from the orbital sinus, and serum was isolated by centrifugation of blood. Paraffin-embedded lung tissue sections were stained with H&E or mucicarmine for histological examination.
GXM levels and clearance. GXM clearance experiments were performed as described (34). Briefly, parental and NOS2-/- mice were treated with mAb i.p. 24 h before i.v. administration of GXM (50 µg). Blood was obtained from the orbital sinus immediately before mAb administration and 2, 6, 23, 35, 59, and 93 h after i.v. GXM administration. Serum was isolated by centrifugation of blood. Mice were sacrificed by cervical dislocation, and organs were removed immediately. Organs were homogenized (Ultra Turrax T25 homogenizer; Janke and Kunkel, Staufen, Germany), and organ homogenate and serum were treated with proteinase K (1 mg/ml, 1 h, 37°C). GXM-capture ELISA was used to detect GXM in the serum, lung, liver, spleen, and kidney of mice, as previously described, except that mAb 2D10 was used for capture and mAb 2H1 was used for detection (35).
Serum nitrite levels. Parental-F1 mice were fed a chemically defined amino acid diet (L-arginine, nitrate and nitrite free; Ziegler Brothers, Gardner, PA). mAb 2H1 and control were administered 24 h before infection IT with 106 C. neoformans. Mice were bled at various intervals from the retroorbital sinus, and serum nitrite was measured using Griess reagent (1% sulfanilamide, 0.1% N-1-napthylethylenediamine, 2.5% phosphoric acid) after reducing nitrate to nitrite with P. oleovorans nitrate reductase, as described (33). The concentration of serum nitrite was calculated from the absorbance at 540 nm using a standard linear curve (0–250 µM).
Cytokine and chemokine studies.
Parental and NOS2-/- mice (6–8 wk old) were
infected, as described above. Experimental groups were given purified
mAb 2H1 or PBS 24 h before infection with yeast. Sham-infected
groups were given mAb 2H1 or PBS 24 h before IT administration of
PBS. Mice were sacrificed at day 14 postinfection, and the right lungs
were homogenized in 2 ml PBS in the presence of protease inhibitors
(Complete Mini; Boehringer Mannheim). The homogenates were centrifuged
at 6000 x g for 10 min to remove cell debris, and the
supernatant was frozen at -80°C until tested. The supernatants were
assayed for concentrations of IL-2, IL-4, IL-6, IL-10, IL-12p70,
monocyte chemoattractant protein (MCP)-1, and macrophage-inflammatory
protein (MIP)-1 using ELISA kits (BD PharMingen (San Diego, CA) and
R&D Systems (Minneapolis, MN)). The detection limits of cytokine assays
are 3.1 pg/ml for IL-2, 7.8 pg/ml for IL-4, 15.6 pg/ml for IL-6 and
TNF-, 31.3 pg/ml for IL-10 and IFN-, and 62.5 pg/ml for IL-12
p40, as stated by the manufacturer. The detection limits of the
chemokine assays are 4.7 pg/ml for MIP-1 and 15.6 pg/ml for MCP-1,
as determined by the manufacturer.
Endotoxin precautions. For the cytokine and cellular response experiments, great care was taken to avoid the contamination of endotoxin. One person, in a laminar flow hood, did all work involving purification of mAb reagents and handling of reagents. Solutions were made with endotoxin-free water or PBS. Extensive use was made of disposable pyrogen-free plastic ware, pipettes, pipet tips, microcentrifuge tubes, etc. Endotoxin concentration in mAb solutions measured by Limulus amebocyte assay (BioWhittaker, Walkersville, MD) was below the limit of detection of the assay.
Preparation of lung leukocytes. Parental and NOS2-/- mice were given mAb and infected, as described above. At day 14 postinfection, lungs were excised, minced, homogenized using a sterile 70-µm nylon mesh (BD Biosciences), and digested for 60 min using 10 ml/lung digestion buffer: RPMI 1640, 10% FCS, 1 mg/ml collagenase (Boehringer Mannheim), and 30 µg/ml DNase I (Sigma-Aldrich). The total cell suspension was collected by centrifugation; erythrocytes were lysed, as described above.
Cell staining and analysis. Neutrophils, eosinophils, lymphocytes, and monocytes/macrophages were visually counted from Giemsa-stained samples of lung cell suspensions centrifuged onto glass slides (Cytospin; Thermo Shandon, Pittsburgh, PA). A total of 200–400 cells was counted from randomly chosen high-power microscope fields for each sample. For FACS analysis, lung leukocytes (106) were stained for 30 min on ice with 100 µl of one of the following Abs diluted in staining buffer: 2 µg/ml R-PE-labeled anti-CD45, 5 µg/ml FITC-labeled anti-mouse CD4, 2 µg/ml CyChrome-labeled anti-mouse CD8, 5 µg/ml FITC-labeled anti-mouse CD19, and 5 µg/ml FITC-labeled anti-mouse MAC-3 (all mAbs from BD PharMingen). The samples were washed twice in staining buffer and fixed in 1% paraformaldehyde. Stained samples were stored in the dark at 4°C until analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest (BD Biosciences) software. Live cells were gated as judged from forward and side laser scatter and CD45+ cells. Controls consisted of isotype-matched irrelevant Abs.
Statistics
All data were analyzed by the Student t test, Kruskal-Wallis test (Primer; McGraw-Hill, New York, NY), and log rank analysis (Sigmastat, Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We investigated the ability of alveolar macrophages isolated from
parental and NOS2-/- mice to phagocytose and
kill C. neoformans. There were no differences in
phagocytosis of C. neoformans by parental and
NOS2-/- alveolar macrophages
(p = 0.82). The phagocytic indices were
significantly higher in the mAb 2H1-treated groups for parental and
NOS2-/- alveolar macrophages in both 2- (data
not shown) and 4-h assays (Fig. 1
A). Incubation of parental
and NOS2-/- alveolar macrophages with C.
neoformans 24067 resulted in a significant reduction in CFU after
18 (data not shown) and 24 h (Fig. 1B). Alveolar
macrophages from parental mice were more effective at inhibiting
C. neoformans than NOS2-/- alveolar
macrophages. To demonstrate that parental alveolar macrophages produced
NO during the killing assay, nitrite levels were measured.
Administration of mAb 2H1 enhanced production of NO correlating with
reduction of CFU (Fig. 1C). As anticipated, NO was not
detected from NOS2-/- alveolar macrophages
during the killing assay (data not shown).
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Survival.
Parental mice infected with C. neoformans survived longer
than NOS2-/- mice (median survival of 31 and 16
days, respectively; p < 0.001) (Fig. 2). Administration of mAb 2H1 (IgG1)
prolonged survival of C. neoformans-infected parental mice
(median survival of 85 days; p < 0.05) relative to
parental control mice, whereas administration of mAb 2H1 did not
prolong survival of NOS2-/- mice (median
survival of 17 days; p = 0.938) (Fig. 2). The
experiment was terminated on day 107, at which time all surviving mice
(three parental control, six parental mAb 2H1-treated, and one mAb
2H1-treated NOS2-/- mice) had detectable lung
CFU, indicating chronic infection. In summary, mAb administration
before infection prolonged survival in parental mice given lethal
C. neoformans infection, but not in
NOS2-/- mice.
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. At
day 14, the lung fungal burden in parental mice was significantly
higher than in NOS2-/- mice
(p < 0.05), but there were no differences in
brain fungal burden (Table I). Administration of mAb 2H1 significantly
reduced lung CFU in parental mice compared with parental control mice
(p = 0.014). To a lesser extent, administration
of mAb 2H1 reduced lung CFU in NOS2-/- mice
(p = 0.223).
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A). In contrast, the lungs of
parental mice treated with mAb 2H1 exhibited an intense granulomatous
inflammation composed of macrophages and epithelioid cells (Fig. 3B). Perivascular cuffs composed of lymphocytes and
polymorphonuclear leukocytes were also present. Histological
examination revealed a different inflammatory response in the lungs of
parental and NOS2-/- mice (Fig. 3, C
and D). A mixed inflammatory infiltrate composed of
polymorphonuclear leukocytes (neutrophils and eosinophils),
lymphocytes, macrophages/monocytes, and epithelioid cells was present
in the perivascular spaces and parenchyma. Mucin staining
demonstrated both extracellular and intracellular C.
neoformans (Fig. 3, insets). There were no differences
in the type and the extent of inflammation in mAb 2H1-treated
NOS2-/- mice compared with
NOS2-/- control mice.
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A). Accordingly, we found no
differences in the total number of cells recruited to the lung of
parental and NOS2-/- mice after infection (Fig. 4B). On day 14, FACS analysis of lung cells showed similar
percentage of lymphocytes and macrophages in the inflammatory
infiltrate of parental and NOS2-/- mice (Fig. 4C). However, analysis of granulocyte composition revealed
significant differences in the proportion of eosinophils and
neutrophils in the inflammatory response of parental and
NOS2-/- mice. There were higher percentages of
eosinophils, irrespective of mAb treatment, in
NOS2-/- mice compared with parental mice
(p = 0.001) (Fig. 4D). In addition,
NOS2-/- mice exhibited higher percentage of
macrophages/monocytes compared with parental mice
(p = 0.025), but no significant differences
were seen between mAb 2H1-treated parental and
NOS2-/- mice (p =
0.473). In contrast, parental mice contained higher percentage of
lymphocytes and neutrophils compared with
NOS2-/- mice (p <
0.001, for both). There were no differences in the percentage of
lymphocytes and neutrophils between mAb 2H1-treated parental and
NOS2-/- mice (p = 0.262
and p = 0.098) (Fig. 4D).
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). In contrast, infected NOS2-/- mice
exhibited significantly higher serum GXM levels compared with parental
mice, irrespective of Abtreatment. Given the significantly higher serum
GXM levels in NOS2-/-, we evaluated the
clearance and distribution of GXM Ag in the presence and absence of mAb
2H1 in NOS2-/- and parental mice. The overall
pattern of GXM clearance and distribution was similar in
NOS2-/- and parental mice, and comparable with
that reported previously (34). Serum GXM was rapidly
cleared in both NOS2-/- and parental mice given
mAb 2H1 and deposited in liver and spleen (Fig. 5).
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C. neoformans-infected parental mice were bled at
various times postinfection, and nitrite concentration in the serum was
measured as described (33). Production of nitrate/nitrite
correlates with in vivo generation of reactive nitrogen intermediates
(33). In the first 8 days postinfection, nitrite levels
were significantly higher in mAb 2H1-treated parental mice compared
with parental control mice (Fig. 6). From
day 14 to day 28 postinfection, serum nitrite levels continued to
increase but were not affected by Ab treatment (data not shown).
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To better understand the immune response in
NOS2-/- andparental mice, in the presence
and absence of Ab, cytokine andchemokine expression were examined.
Preliminary studies using the RNase protection assay revealed
differences in cytokine/chemokine expression at day 14, but not at days
1 and 7 postinfection (data not shown). Hence, we proceeded to measure
cytokine protein levels at day 14 of infection. At day 14
postinfection, NOS2-/- mice produced
significantly higher pulmonary concentrations of IFN- than parental
mice, irrespective of mAb 2H1 treatment (Fig. 7, upper panel). MCP-1 and
MIP-1 are induced in the lungs of NOS2-/-
and parental mice infected with C. neoformans (Fig. 7, middle and lower panels).
NOS2-/- mice produced significantly higher
concentrations of MCP-1 and MIP-1 than parental mice, which is
consistent with the intense inflammatory response seen in these mice.
Notably, mAb significantly decreased IFN-, MCP-1, and MIP-1
(p = 0.02) in the lungs of
NOS2-/- mice. Additional cytokines were
measured (IL-2, IL-4, IL-6, IL-10, IL-12, and TNF-), but no
differences were noted between parental and
NOS2-/- mice (Table II). However, for some cytokines, the
levels measured after infection were lower than in sham-infected mice
(Table II). IL-2 and TNF- were lower in lungs of infected
NOS2-/- than in sham-infected
NOS2-/- mice. Similarly, IL-4 and IL-10 were
lower in the lungs of infected parental mice than in sham-infected
mice. Ab administration had little or no effect on the levels of these
cytokines during infection, except for IL-4, which was increased in
parental mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NO is a critical microbicidal molecule (17) that is also a mediator of immunopathology (17, 43, 44), possibly through regulatory effects on Th1 cells (45). Histological studies revealed that the appearance of an inflammatory response in the lungs of NOS2-/- mice was different from the inflammatory response found in the lungs of parental mice. However, there was no difference in lung weights or total inflammatory cells in the lungs of NOS2-/- and parental mice. Similarly, lungs from parental mice treated with Ab had similar lung weights and total inflammatory cells compared with parental mice not receiving Ab. The finding of differences in histological appearance for lungs that contained the same number of inflammatory cells suggests that similar numbers of cells migrate into the lung in response to infection, but that these achieve different tissue distribution. Qualitative analysis of the lung infiltrate revealed similar numbers of CD4, CD8, and B lymphocytes. However, we noted that the number of eosinophils in lungs from NOS2-/- mice was significantly higher than parental mice, irrespective of Ab administration. Eosinophils are recruited to inflammatory sites to a variety of infections (46, 47, 48, 49). Eosinophilic granules contain polycationic proteins that are highly toxic to several pathogens including C. neoformans (49), as well as mammalian cells including respiratory epithelial cells (50, 51). Eosinophils are potentially toxic to host tissues because of their toxic granule contents and their ability to generate superoxide radicals and leukotrienes (reviewed in Ref. 52). In fact, eosinophils can damage lung tissue by a novel mechanism that involves brominating proteins (53). The marked eosinophilia in the inflammatory response of NOS2-/- mice may account for the seemingly paradoxical effects of shorter survival as a result of increased lung damage and lower CFU through the powerful antifungal action of eosinophil granules.
The differences in inflammatory response in
NOS2-/- and parental mice suggest the
involvement of NO in the regulation of the inflammatory response to
C. neoformans. In this regard, we found greater production
of IFN-, MIP-1, and MCP-1 in the lungs of C.
neoformans-infected NOS2-/- compared with
parental mice, suggesting that NO may function as an important
regulator of the inflammatory response through its effect on the
expression of certain cytokines. Recently, Aguirre et al.
(37) reported no differences in IFN- production in
bronchoalveolar lavage fluid between NOS2-/-
and parental mice during IT C. neoformans infection. The
discrepancy between our observation and that of Aguirre et al.
(37) is probably due to methodological differences,
because we measured total lung cytokine level, while they studied the
concentration in alveolar lavage. In addition, we measured lower levels
of IL-2 and TNF- in NOS2-/--infected mice
than in sham-infected mice, a phenomenon that was not observed for
parental mice. These observations may reflect NO-mediated effects on
cytokine expression and inflammation, and are consistent with in vitro
studies demonstrating that NO can inhibit both mitogen and Ag-induced
proliferation of lymphocytes (54, 55).
Ab administration prolonged survival in parental mice, but not in NOS2-/- mice. This result is consistent with our prior studies that showed that passive administration of IgG1 mAb prolonged survival of C. neoformans-infected mice (13, 28, 29, 30). Serum nitrite levels were significantly higher in Ab-treated parental mice in the days immediately following cryptococcal infection, whereas parental control mice manifested a reduction in serum nitrite levels after infection. Because C. neoformans reduces macrophage production of NO in vitro (56), the reduced levels of serum nitrite in parental control mice may be an in vivo correlate of this phenomenon. The higher levels of serum nitrite in Ab-treated mice could also reflect enhanced NO synthesis due to FcR cross-linking (57). The observation that Ab administration prevents a decrease in serum nitrite suggests a new mechanism of action for specific Ab against C. neoformans that could have profound effects on host defense given the protean effects of nitrogen-related radicals on immune function (17). We also noted that Ab-treated parental mice had higher IL-4 levels. Although we did not measure a cellular correlate for higher IL-4 levels in parental mice, it is conceivable that higher levels of this inflammation-inhibiting cytokine contributed to prolonged survival as a result of reduced lung damage.
The most striking result of our study was the observation that Ab
prolonged survival in parental, but not NOS2-/-
mice, despite producing a modest reduction in
NOS2-/- lung fungal burden. To our knowledge,
Ab efficacy has not been studied in NOS2-/-
mice against any pathogen. The cytokine response in
NOS2-/- and parental mice was different such
that infection increased the pulmonary level of IFN- in
NOS2-/- mice, but reduced it in parental mice.
Furthermore, Ab administration had different effects on cytokine
production in NOS2-/- and parental mice,
reducing IFN- in NOS2-/- mice and increasing
it in parental mice. These observations suggest that when the stimulus
for increased lung IFN- is Ab, passive Ab administration is
effective in prolonging survival. However, when the stimulus for high
levels of lung IFN- is infection, in the setting of genetic
deficiency of NOS2, passive Ab is not effective in prolonging survival.
Thus, NO may exert its effect on Ab-mediated efficacy through effects
on the regulation of IFN-. This result is consistent with reports
that NO has protective effects against T cell-dependent shock induced
by Toxoplasma gondii (58), staphylococcal
enterotoxin B (59, 60), and L. major
(61) by down-regulating IFN- and TNF- production. In
the absence of NO, Ab may deliver an inhibitory signal, which reduces
IFN- production in NOS2-/- mice. This is
consistent with previous studies demonstrating antiinflammatory
activity of Ig through FcRs (62).
Ab-treated NOS2-/- mice had very high serum levels of GXM relative to Ab-treated parental mice, despite lower organ fungal burden and Ab-mediated clearance of immune complexes with deposition of polysaccharide Ag in liver, as reported in prior studies of normal animals (34). Because Ab-mediated clearance of GXM in NOS2-/- mice appears to be intact, the mechanism responsible for the much higher levels of GXM in Ab-treated NOS2-/- mice is unknown. Potential mechanisms include the existence of an unidentified Ag clearance defect in NOS2-/- mice, or enhanced polysaccharide production by C. neoformans in inflammatory tissues that are devoid of NO. Remarkably, NOS2-/- mice with C. neoformans infection had intense inflammation despite high levels of serum GXM. Higher cytokine/chemokine levels in NOS2-/- mice could have mitigated any antiinflammatory properties or other effects that GXM may have, and it is conceivable that, in certain hosts, GXM itself is proinflammatory (63, 64). Thus, irrespective of its effects on CFU reduction and/or the modulation of inflammatory responses, Ab-mediated clearance of GXM may be pivotal and required for Ab efficacy against C. neoformans.
Our study explored the mechanisms responsible for Ab efficacy against
C. neoformans, confirmed an important role for NOS in host
defense against experimental murine cryptococcal infection, and
established that Ab efficacy was dependent on NO production.
Furthermore, our results strongly suggest that NO is an important
regulator of immune responses that can affect Ab efficacy. IFN-
appears to be beneficial or harmful depending on its tissue level and
is likely to have a critical role in the mechanisms that determine
either early death or survival in NOS2-/- and
parental mice, respectively. The emerging picture indicates that Ab
exerts profound effects on the inflammatory response by mechanisms that
include changes in IFN-, serum polysaccharide levels, and nitrite
production, in addition to the more classical role of Ab as an opsonin.
The requirement for NO in host defense and Ab function was illustrated
by the dramatic differences observed in Ab efficacy, inflammatory
response, and cytokine production in NOS2-/-
and parental mice infected with C. neoformans in the
presence and absence of specific Ab. Our results provide additional
evidence for the interdependency of the humoral and cellular arms of
the immune system, and suggest a need to carefully dissect mechanisms
of Ab function based on its effect on the expression of immune
mediators. The requirement for NO in Ab-mediated protection against
C. neoformans identifies a new variable in Ab efficacy that
may also be applicable to other pathogens.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arturo Casadevall, Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: casadeva{at}aecom.yu.edu
3 Abbreviations used in this paper: NOS2, inducible NO synthase; GXM, glucuronoxylomannan; IT, intratracheal(ly); MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein.
Received for publication August 6, 2001. Accepted for publication January 25, 2002.
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interferon-induced nitric oxide in pulmonary clearance of Cryptococcus neoformans. Am. J. Respir. Cell Mol. Biol. 13:116.[Abstract]
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M. Marks, T. Burns, M. Abadi, B. Seyoum, J. Thornton, E. Tuomanen, and L.-a. Pirofski Influence of Neutropenia on the Course of Serotype 8 Pneumococcal Pneumonia in Mice Infect. Immun., April 1, 2007; 75(4): 1586 - 1597. [Abstract] [Full Text] [PDF]
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F. L. Wormley Jr., J. R. Perfect, C. Steele, and G. M. Cox Protection against Cryptococcosis by Using a Murine Gamma Interferon-Producing Cryptococcus neoformans Strain Infect. Immun., March 1, 2007; 75(3): 1453 - 1462. [Abstract] [Full Text] [PDF]
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L.-A. Pirofski Of Mice and Men, Revisited: New Insights into an Ancient Molecule from Studies of Complement Activation by Cryptococcus neoformans. Infect. Immun., June 1, 2006; 74(6): 3079 - 3084. [Full Text] [PDF]
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 Y.-T.A. Teng Protective and Destructive Immunity in the Periodontium: Part 1--Innate and Humoral Immunity and the Periodontium Journal of Dental Research, March 1, 2006; 85(3): 198 - 208. [Abstract] [Full Text] [PDF]
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 J. Rivera and A. Casadevall Mouse Genetic Background Is a Major Determinant of Isotype-Related Differences for Antibody-Mediated Protective Efficacy against Cryptococcus neoformans J. Immunol., June 15, 2005; 174(12): 8017 - 8026. [Abstract] [Full Text] [PDF]
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 S. A. Miles, S. M. Conrad, R. G. Alves, S. M.B. Jeronimo, and D. M. Mosser A role for IgG immune complexes during infection with the intracellular pathogen Leishmania J. Exp. Med., March 7, 2005; 201(5): 747 - 754. [Abstract] [Full Text] [PDF]
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J. Rivera, O. Zaragoza, and A. Casadevall Antibody-Mediated Protection against Cryptococcus neoformans Pulmonary Infection Is Dependent on B Cells Infect. Immun., February 1, 2005; 73(2): 1141 - 1150. [Abstract] [Full Text] [PDF]
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A. Casadevall and L.-a. Pirofski New Concepts in Antibody-Mediated Immunity Infect. Immun., November 1, 2004; 72(11): 6191 - 6196. [Full Text] [PDF]
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 R. C. Reddy, V. G. Keshamouni, S. H. Jaigirdar, X. Zeng, T. Leff, V. J. Thannickal, and T. J. Standiford Deactivation of murine alveolar macrophages by peroxisome proliferator-activated receptor-{gamma} ligands Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L613 - L619. [Abstract] [Full Text] [PDF]
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C. P. Taborda, J. Rivera, O. Zaragoza, and A. Casadevall More Is Not Necessarily Better: Prozone-Like Effects in Passive Immunization with IgG J. Immunol., April 1, 2003; 170(7): 3621 - 3630. [Abstract] [Full Text] [PDF]
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G. M. Cox, T. S. Harrison, H. C. McDade, C. P. Taborda, G. Heinrich, A. Casadevall, and J. R. Perfect Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages Infect. Immun., January 1, 2003; 71(1): 173 - 180. [Abstract] [Full Text] [PDF]
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