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Published ahead of print on July 10, 2003, doi:10.1165/rcmb.2003-0156OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 109-117, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0156OC

Role of Interleukin-4 in Resistance to Cryptococcus neoformans Infection

Rebecca Blackstock and Juneann W. Murphy

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Address correspondence to: Rebecca Blackstock, Ph.D., Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190. E-mail: becky-blackstock{at}ouhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of interleukin (IL)-4 in cryptococcal disease was studied in IL-4 knockout (IL-4KO) and wild-type (WT) mice infected with Cryptococcus neoformans isolates that vary widely in their virulence. Delayed-type hypersensitivity responses were reduced in IL-4KO mice following primary infection with either isolate. Splenic T helper 1 (Th1) cytokine responses were increased in the IL-4KO mice infected with the weakly virulent isolate (184A) but did not change during infection with the highly virulent isolate (NU-2). Th2 cytokine responses (IL-5, IL-10) were downregulated in the IL-4KO mice infected with either isolate. Survival after primary infection with either isolate was not influenced by the absence of IL-4. Fewer colony-forming units were found in the lungs of 184A-infected, IL-4KO mice as compared to WT mice, suggesting that some immunity had developed. IL-4KO mice, primed with small doses of cryptococcal antigen (CneF), had significantly enhanced delayed-type hypersensitivity responses after intravenous infection with 184A and were more resistant to infection compared with WT mice. Increased expression of IL-5 with decreased interferon-{gamma} contributed to the inability of primed WT mice to resist infection with 184A. Enhanced immunity in the primed IL-4KO mice was reflected in a more moderate increase in IL-5 and IL-10 with maintenance of interferon-{gamma} levels.

Abbreviations: colony-forming units, cfu • cell-mediated immunity, CMI • cryptococcal culture filtrate antigen, CneF • delayed-type hypersensitivity, DTH • enzyme-linked immunosorbent assay, ELISA • fetal bovine serum, FBS • Hanks' balanced salt solution, HBSS • interferon-{gamma}, IFN-{gamma} • interleukin, IL • IL-4–deficient mice, IL-4KO • sterile physiological saline solution, SPSS • T helper 1, Th1 • T helper 2, Th2 • wild-type, WT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cryptococcus neoformans, a yeast-like organism found worldwide in soils, causes infection in humans after being inhaled. If the organism survives in the lung, it may disseminate to other tissues via the blood stream (1). C. neoformans has a predilection for the central nervous system, so most frequently the disease is first recognized in humans as a meningoencephalitis (2). Since the beginning of the AIDS epidemic, C. neoformans has become important as one of the primary causes of death in patients with AIDS (1). This clinical finding correlates with results in experimental models showing that cell-mediated immunity (CMI), characterized by activation of CD4+ T-helper 1 (Th1) cells, which produce interferon (IFN)-{gamma}, is essential for protection against the organism or for complete recovery from cryptococcosis (3–7).

Induction of activated Th1 cells, which are the key players in the CMI response, occurs when Th1-associated cytokines (interleukin [IL]-2, IL-12, and IFN-{gamma}) are induced (8–11). Th2 cells, which secrete IL-4, IL-5, and IL-10, have been shown to inhibit Th1 cell induction (12, 13). Some reports suggest that IL-4 may be needed to establish and/or maintain Th1-mediated CMI responses against eukaryotic pathogens (14, 15). For example, IL-4 is needed for an adequate Th1 response to Candida albicans (14), and IL-4–deficient mice are more susceptible to Toxoplasma gondii than are their wild-type (WT) counterparts (15). Considering these findings and the fact that protection against both C. albicans and T. gondii is mediated by a CMI response characterized by the production of IFN-{gamma} (16), we hypothesized that some IL-4 may be needed for optimal induction of a protective, anticryptococcal CMI response.

We decided to test our hypothesis by examining survival, Th1 and Th2 cytokine responses, and delayed-type hypersensitivity (DTH) responses in WT and IL-4 knockout (IL-4KO) mice infected with a weakly virulent or highly virulent isolate of C. neoformans. We previously reported that cryptococci of varying pathogenic potential for mice induce cytokines that differ in quality and in the kinetics of their induction (17). DTH analyses were included in our study because the DTH reaction is known to correlate with protective immunity when combined with mononuclear infiltrates and elevated IFN-{gamma} levels in DTH reaction sites (18). In addition, high IFN-{gamma} levels without DTH responses do not provide protective immunity to mice infected with C. neoformans (19, 20). Results of these studies revealed that IL-4 was needed for development of strong DTH responses after primary infection with both cryptococcal isolates, and IL-4 deficiency did not increase survival of mice undergoing a primary infection with either isolate. On the other hand, Th1 cytokine responses detected 30 d after infection with the weakly virulent cryptococcal isolate were significantly increased under IL-4–deficient conditions, suggesting that the IL-4–deficient mice were beginning to respond to their infection. The poor outcomes of mice infected with the highly virulent cryptococcal isolate could not be attributed to induction of an IL-4 response during infection. IL-4 deficiency did increase DTH reactions and survival of IL-4KO mice that were primed with cryptococcal antigen before infection with the weakly virulent isolate. This was attributed to lower Th2 responses in the primed IL-4KO mice preventing downregulation of the IFN-{gamma} response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wild-type, C57BL/6J (WT) and C57BL/6J-Il4tm1Cgn (IL-4KO) female mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The animals were used in experiments when they were 14–16 wk of age. Mice were housed in the animal facility at the University of Oklahoma Health Sciences Center. The facility is approved by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).

Reagents
RPMI-1640, penicillin-streptomycin, L-glutamine, sodium pyruvate, essential vitamins, and nonessential amino acids were purchased from GIBCO BRL (Grand Island, NY). Fetal bovine serum (FBS) was purchased from HyClone (Ogden, UT). Concanavalin A and HEPES were obtained from the Sigma Chemical Co (St. Louis, MO). Recombinant mouse IL-2 was purchased from Collaborative Biomedical Products (Bedford, MA). Recombinant IFN-{gamma} was obtained from Genzyme (Cambridge, MA). Recombinant IL-4 was generously provided by Sterling Winthrop (Malvern, PA). Recombinant IL-5 and paired monoclonal antibodies used for mouse cytokine enzyme-linked immunosorbent assays (ELISA) were purchased from PharMingen (San Diego, CA). IL-10 levels were measured using a PharMingen IL-10 OptEIA kit.

Maintenance of Entodoxin-Free Conditions
To assure that the presence of endotoxin did not influence our experimental results, all experiments were performed under conditions that would minimize endotoxin contamination. Whenever possible, sterile plasticware was used and any glassware used was heated for 3 h at 180°C. All reagents contained < 8 pg of endotoxin/ml (minimal detectable level) when tested using the Limulus amoebocyte lysate assay (Whittaker Bioproducts, Inc., Walkersville, MD).

Fungal Strains
Cryptococcal isolate NU-2 was originally isolated from the spinal fluid of a patient with cryptococcal meningitis at the University of Nebraska Medical Center. Isolate 184A was originally obtained from Dr. L. Friedman, Tulane University Medical School (New Orleans, LA). Both organisms are serotype A. Isolate NU-2 is highly virulent for mice while isolate 184A is classified as weakly virulent for mice. Both isolates were maintained in the laboratory by growth on Sabouraud's dextrose agar.

Preparation of Cryptococcal Antigen
A cryptococcal culture filtrate antigen (CneF) prepared from cryptococcal isolate 184A was described previously (3). The lot used in this investigation had a protein content of 243 µg/ml as determined by the bicinchoninic assay (Pierce Chemical Co., Rockford, IL) and a carbohydrate content of 3.2 mg/ml as determined by the phenol-sulfuric assay (21). When this lot of CneF was tested with the Limulus assay, endotoxin levels were less than 0.1 ng of endotoxin/ml (lower limit of detection).

Intratracheal Infections
Cryptococci were grown on Sabouraud's dextrose agar for 72 h. The cells were harvested from the surface of the agar using sterile physiologic saline solution (SPSS) and washed three times with SPSS. The cells were counted on a hemocytometer and adjusted to a concentration of 4 x 106 per ml. WT and IL-4KO mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg). The trachea of each mouse was exposed surgically and a 22-gauge angiocatheter was inserted into the trachea. Twenty-five microliters of cryptococcal suspension, containing 104 or 105 cryptococci, was injected into the angiocather tube using a sterile Hamilton syringe. This was followed by injection of 50 µl of air to clear the cryptococci from the angiocatheter tube and deposit the fungal organisms in the lung. Sham controls were inoculated with 25 µl of SPSS.

Experimental Protocol
For studies of primary infection, mice were infected intratracheally with 1 x 105 NU-2 or 184A suspended in 25 µl of SPSS or they were infected intravenously with 1 x 105 184A. Sham-infected control mice were inoculated intratracheally with 25 µl of SPSS or intravenously with SPSS. Spleen cells were removed at various times after infection and studied for their ability to secrete cytokines when cultured with CneF. Animals studied for their secondary immune response to cryptococcal antigen were primed by injection of 30 µl of CneF into the hind footpad on Day 0 (3). The primed mice were challenged with 1 x 105 184A administered intravenously 30 d later. DTH reactions were determined on the day of infection and 15 d after infection. These same animals were followed to determine their survival to challenge with 184A. In a separate experiment mice were primed and infected intravenously with 1 x 105 184A blastoconidia 30 d later. Spleen cells from these mice were removed on the 12th day of infection and cultured with or without CneF. Supernatant fluids were assayed by ELISA to determine the levels of Th1 and Th2 cytokines secreted by the spleen cells.

Elicitation of the Anticryptococcal DTH Response
Hind footpads of mice were measured with a gauge micrometer (Mitutoyo, Aurora, IL). The right hind footpad was subsequently injected with 30 µl of CneF, and the left hind footpad was injected with 30 µl SPSS. Twenty-four hours later, the footpads were measured a second time. The increase in footpad thickness was calculated as the difference in swelling between the 0- and 24-h measurements. Specific DTH reactivity was calculated as the difference between the swelling of the SPSS-injected footpad and the swelling of the CneF-injected footpad.

In Vitro Stimulation of Cytokine Synthesis by Spleen Cells
Spleens were harvested from experimental mice at various times after infection with C. neoformans strains NU-2 or 184A. Single cell suspensions of the spleens were prepared by pressing the cells through a 60-mesh wire screen into sterile Hanks' balanced salt solution (HBSS) containing 3% FBS (HBSS-FBS). The cells were washed three times in HBSS-FBS and resuspended in Bretcher's medium (RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 25 mM L-glutamine, 1 mM sodium pyruvate, 1% essential vitamins, 1% nonessential amino acids, and 10% FBS). The spleen cells were cultured at a concentration of 5 x 106 per ml with CneF at a final concentration of 1:8. Separate samples of 5 x 106 cells/ml were cultured without stimulation (background control) or were stimulated with concanavalin A (10 µg/ml) as a positive control (data not shown). The cultures were incubated at 37°C in an atmosphere of 5% CO2. Supernatant fluids were collected at 24 h when IL-2 levels were analyzed and at 48 h when levels of all other cytokines were studied.

Quantitation of Cytokine Levels in Culture Supernatants
Enzyme-linked immunosorbent assays for the detection of IL-2, IL-4, IL-5, and IFN-{gamma} were constructed using commercially available paired monoclonal antibodies. IL-10 levels were measured using a PharMingen OptEIA IL-10 assay kit. Minimal levels of detection were: IL-2 = 4 pg/ml, IL-4 = 6.25 pg/ml, IL-5 = 25 pg/ml, IL-10 = 33.3 pg/ml, and IFN-{gamma} = 128 pg/ml.

Determination of Survival after Infection with C. neoformans
The virulence of NU-2 or 184A during primary infection in WT or IL-4KO mice was determined after infecting mice intratracheally with 1 x 104 or 1 x 105 NU-2 or 184A as determined by hemocytometer counts. Colony-forming units (cfu) in the inoculum were determined by dilution plate counting and are reported for individual experiments. In designated experiments, virulence of 184A in mice was determined after intravenous infection with 1 x 105 184A. The infected animals were observed daily for deaths to determine survival times.

Statistical Analysis
Analysis of the statistical difference between two groups was evaluated with Student's t test and the F test for analysis of variance. In experiments where there was a significant difference in the variances of the groups, the t test was used with Welch's correction. Survival data were analyzed using Kaplan-Meier survival plots followed by log rank tests. Data with a P value of 0.05 or less were considered to be significant. All observations were made at least two times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DTH Reactions during Primary Infection of WT and IL-4KO Mice with Weakly Virulent and Highly Virulent Cryptococcal Isolates
Our initial investigations into the role that IL-4 might play in cryptococcal infection were designed to determine if mice that were deficient in their ability to make IL-4 could mount a CMI response after infection with a highly virulent (NU-2) or weakly virulent (184A) cryptococcal isolate. For this evaluation, WT or IL-4KO mice were infected by intratracheal instillation of 1 x 105 C. neoformans cells. Control (uninfected) mice of both strains received the same volume of SPSS intratracheally (sham treated). Twenty days later, infected and control mice were tested for their DTH reactivity to the CneF skin test antigen. The results of a typical experiment are seen in Figure 1. DTH responses were significantly reduced in infected IL-4KO mice whether they were infected with the highly virulent (NU-2) or weakly virulent (184A) cryptococcal isolate, suggesting that IL-4 may be needed for early development of DTH reactivity that has been associated with protective immunity in this disease.



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  		Figure 1. Delayed-type hypersensitivity responses in WT (open bars) and IL-4KO (hatched bars) mice infected with cryptococcal isolates 184A or NU-2. Mice were infected intratracheally with 1 x 105 NU-2 or 184A. DTH responses to cryptococcal CneF antigen were determined on the 20th day after infection. Data from five mice per group are shown as the mean ± SEM of the increase in paw thickness expressed in inches (in).

 
Th1 Cytokine Responses of CneF-Stimulated Spleen Cells Harvested during Primary Infection of WT and IL-4KO Mice with Weakly Virulent and Highly Virulent Cryptococcal Isolates
Considering that DTH reactivity was decreased in the IL-4KO mice, we anticipated that there would be a change in cytokine production in IL-4KO as compared with WT mice. Mice were infected with 1 x 105 C. neoformans NU-2 or 184A intratracheally, or were treated with SPSS by intratracheal instillation. At various times after infection, spleen cells were removed and cultured in the presence or absence of CneF antigen. The spleen cell supernatant fluids were evaluated for their levels of Th1 (IL-2 and IFN-{gamma}) or Th2 (IL-4, IL-5, IL-10) cytokines by ELISA.

At Days 12 and 20 after 184A infection, IL-2 levels produced by CneF-stimulated spleen cells were significantly higher in the IL-4KO mice than in the WT mice (Figure 2). During NU-2 infection IL-2 responses were quite low, and no significant differences in IL-2 levels were detected at any time point when the responses of antigen-stimulated spleen cells obtained from IL-4KO mice were compared with similar results obtained from spleen cell culture supernatants derived from WT mice. IL-2 levels decreased over time in the spleen cell cultures of NU-2–infected mice, and all of the NU-2–infected mice were dead before Day 30.



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  		Figure 2. Th1 cytokines secreted by spleen cells taken from WT (squares) or IL-4KO (triangles) mice at various times after infection with cryptococcal isolates NU-2 or 184A. Spleen cells were cultured with or without the addition of cryptococcal antigen CneF, and supernatants were harvested 24 h (IL-2) or 48 h (IFN-{gamma}) after the initiation of cultures. IL-2 response of sham WT mice: medium = 8.48 ± 1.86 pg/ml, CneF = 16.9 ± 2.69 pg/ml. IL-2 response of sham IL-4KO mice: medium = 8.58 ± 1.54 pg/ml, CneF = 24.87 ± 3.03 pg/ml. IFN-{gamma} response of sham WT mice: medium = 2.0 ± 2.0 pg/ml, CneF = 12.66 ± 6.51 pg/ml. IFN-{gamma} response of sham IL-4KO mice: medium = 5.65 ± 3.11 pg/ml, CneF = 35.45 ± 14.33 pg/ml. Vertical error bars represent the variation (± SEM). There were five animals in each group.

 
In 184A-infected IL-4KO mice, IFN-{gamma} responses increased with time over the first 30 d of infection (Figure 2). Significant increases in IFN-{gamma} production were detected at Days 24 and 30 after infection in spleen cell cultures derived from IL-4KO mice as compared with similar cultures that were derived from WT mice. IFN-{gamma} responses in spleen cell cultures of WT or IL-4KO mice infected with the highly virulent isolate, NU-2, were very low at all time points, and there were no significant differences between the two groups at any time period during which evaluations were performed (Figure 2).

Th2 Cytokine Responses of CneF-Stimulated Spleen Cells Harvested during Primary Infection of WT and IL-4KO Mice with Weakly Virulent and Highly Virulent Cryptococcal Isolates
IL-4 was not detected in CneF-stimulated spleen cell culture supernatants prepared from infected WT or IL-4KO mice (data not shown). Analysis of IL-5 levels in supernatants from spleen cell cultures stimulated with CneF showed that IL-5 levels were lower in cultures derived from IL-4KO mice as compared with cultures derived from WT mice at all time periods tested after infection with 184A (Figure 3); however, the values were only significantly different at 18 and 30 d after infection. IL-5 levels were significantly decreased in CneF-stimulated spleen cell cultures from NU-2–infected IL-4KO mice when compared with NU-2–infected WT mice on Days 12 and 24 after infection (Figure 3). IL-5 levels in spleen cell cultures from NU-2–infected WT mice were higher early in infection than IL-5 levels in spleen cell cultures of 184A-infected WT mice at the same time periods. However, IL-5 levels tended to decrease after Day 12 in NU-2–infected mice (Figure 3).



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  		Figure 3. Th2 cytokines secreted by spleen cells taken from WT (squares) or IL-4KO (triangles) mice at various times after infection with cryptococcal isolates NU-2 or 184A. Spleen cells were cultured with or without the addition of cryptococcal antigen CneF, and supernatants were harvested 48 h after the initiation of cultures. IL-5 response of sham WT mice: medium = 26.52 ± 1.66 pg/ml, CneF = 22.72 ± 2.18 pg/ml. IL-5 response of sham IL-4KO mice: medium = 30.75 ± 1.85 pg/ml, CneF = 26.92 ± 2.97 pg/ml. IL-10 response of sham WT mice: medium = 0.260 ± 0.1 pg/ml, CneF = 0.299 ± 0.016 pg/ml. IL-10 response of sham IL-4KO mice: medium = 0.268 ± 3.11, CneF = 35.45 ± 14.33 pg/ml. Vertical error bars represent the variation (± SEM). There were five animals in each group.

 
IL-10 levels detected in culture supernatants of spleen cells harvested from WT and IL-4KO mice infected with cryptococcal isolates NU-2 or 184A are shown in Figure 3. IL-4 did not appear to regulate the expression of IL-10 when mice were infected with the weakly virulent isolate, 184A, because there was no significant difference in the level of IL-10 in culture supernatants of spleen cells from the two mouse strains infected with 184A. During an NU-2 infection, there was a significant reduction in the IL-10 response of CneF-stimulated spleen cells of the IL-4KO compared with production by spleen cells of NU-2–infected WT mice on the 12th day after infection. Thereafter, IL-10 levels tended to be lower in spleen cell supernatants derived from IL-4KO mice, but these differences were not statistically significant. As with the other cytokine levels, the overall production of IL-10 during NU-2 infection decreased over the duration of the infection until the mice died.

Survival of WT and IL-4KO Mice after Primary Infection with the Highly Virulent Cryptococcal Isolate, NU-2
The ability of IL-4–deficient and WT mice to resist infection with the highly virulent cryptococcal isolate is shown in Figure 4. Mice were infected intratracheally with 6.75 x 103 cfu NU-2 and observed daily for deaths. In the experiment shown, there was no significant difference in the survival of WT mice infected with NU-2 as compared with IL-4KO mice infected with the same isolate. In fact, the IL-4KO mice appeared to be slightly less resistant to the infection. This observation occurred consistently in repeated experiments but only reached statistical significance in one out of three experiments.



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  		Figure 4. Survival of WT (solid squares) and IL-4KO (open squares) mice infected intratracheally with 6.75 x 103 cfu cryptococcal isolate NU-2. Deaths were recorded daily. There were 10 animals in each group.

 
Cryptococcal cfu in Lungs of WT and IL-4KO Mice after Primary Intratracheal Infection with Cryptococcal Isolate 184A
Because very few mice infected intratracheally with 184A die, we assessed the abilities of IL-4KO and WT mice to clear cryptococcal cells after an intratracheal infection with 1 x 105 blastoconidia of 184A. Twenty days after infection, the lungs, liver and brains were removed, homogenized and serially diluted to determine the number of cryptococcal cfu per organ. At 20 d after infection, IL-4–deficient mice showed a significantly (P < 0.02) enhanced ability to clear the 184A organisms from the lung (2.4 ± 0.7 x 105 cfu/lung) when compared with clearance from the WT animals (3.7 ± 2.4 x 104 cfu/lung). No significant differences were detected in cfu counts from WT and IL-4KO lungs infected with 184A at earlier time points after infection (data not shown). Colony-forming unit determinations from brain and liver on the 20th day of infection revealed a trend toward lower numbers of organisms in the IL-4–deficient animals, but these differences were not statistically significant. No differences in cfu in brain and liver were detected at other time points.

Survival of WT and IL-4KO Mice after Primary Intravenous Infection with the Weakly Virulent Cryptococcal Isolate, 184A
Because intravenous challenge with the weakly virulent cryptococcal isolate will cause death in animals, this challenge route was used to study survival of WT and IL-4KO mice to 184A infection. After primary infection (1.4 x 105 cfu) with 184A by the intravenous route, IL-4KO mice were not more resistant to infection than WT mice based on survival (Figure 5). The IL-4–deficient mice infected with 184A also exhibited a tendency to die faster than the WT controls, but this difference was not statistically significant (Figure 5).



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  		Figure 5. Survival of WT (solid squares) or IL-4KO (open squares) mice infected intravenously with 1.4 x 105 cfu cryptococcal isolate 184A. Deaths were recorded daily and represent the survival curves for nine individual mice per group.

 
DTH Responses in WT and IL-4KO Mice Primed with Cryptococcal Antigen before Infection with the Weakly Virulent Cryptococcal Isolate, 184A
Because IL-4KO mice develop higher Th1 cytokine responses after being stimulated by C. neoformans infection than did WT mice and the IL-4KO mice cleared 184A from their lungs better than WT mice, we decided to examine the effects of priming mice with cryptococcal antigen before infection with 184A. WT and IL-4KO mice were primed by injection of 30 µl of cryptococcal CneF antigen into the hind footpad. Thirty days later the mice were challenged with 1 x 105 184A intravenously. DTH responses were measured on Day 15 of infection. When analyzed for their secondary immune response, infected IL-4KO mice responded to cryptococcal antigen with significantly (P < 0.045) greater DTH reactivity than did WT mice treated in the same manner (Figure 6).



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  		Figure 6. DTH responses in WT and IL-4KO mice primed with cryptococcal CneF antigen before infection with cryptococcal isolate 184A. Mice were primed with 30 µl (open bars) of SPSS or 30 µl CneF (hatched bars). Thirty days later the mice were infected intravenously with 1 x 105 184A. Animals were footpad tested with CneF or saline 15 d after infection. Data from five mice per group are shown as the mean ± SEM expressed in inches (in).

 
Survival of WT and IL-4KO Mice Primed with Cryptococcal Antigen before Infection with the Weakly Virulent Cryptococcal Isolate, 184A
IL-4KO and WT mice were primed with CneF antigen by injection of 30 µl of antigen into the footpad. Thirty days later primed mice and normal control mice were challenged intravenously with 1 x 105 cfu 184A and were observed daily for deaths. When the mice were primed with cryptococcal antigen before infection, IL-4KO mice resisted intravenous infection with 184A much better than did WT IL-4–sufficient mice (Figure 7). This difference was highly statistically significant (P = 0.0029).



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  		Figure 7. Survival of WT (solid squares) or IL-4KO (open squares) mice primed with cryptococcal CneF antigen before infection with cryptococcal isolate 184A. Mice were primed with 30 µl CneF. Thirty days later the mice were infected intravenously with 1 x 105 cfu 184A. Data represent the survival curves for 10 individual mice per group. The two groups are significantly different (P = 0.0029).

 
Cytokine Responses of CneF-Stimulated Spleen Cells of WT and IL-4KO Mice Primed with Cryptococcal Antigen before Infection with the Weakly Virulent Cryptococcal Isolate, 184A
To examine the mechanism responsible for the enhanced resistance of primed IL-4KO mice to intravenous challenge with 184A, mice were primed with cryptococcal antigen and 30 d later were infected intravenously with 184A. Spleen cells were removed on the 14th day after infection. Spleen cells were cultured with or without cryptococcal CneF antigen and cytokines secreted into culture supernatants were measured by ELISA. Compared with CneF-stimulated spleen cell cultures from infected WT mice, antigen-stimulated spleen cells of IL-4KO mice secreted significantly more IFN-{gamma} and less IL-5 (Table 1). The levels of IL-10 secreted by the infected, IL-4 KO spleen cells were also lower than the infected WT spleen cells, but these differences were not statistically significant. Antigen-stimulated spleen cells from primed and infected IL-4KO mice also tended to produce less IL-10 than spleen cells from primed and infected WT mice, but the data were not statistically different. In this instance the overall levels of Th1 cytokines secreted by the primed, infected IL-4KO mice were not different than infected IL-4KO mice that were not primed. However, the level of IL-5 secreted by primed, infected WT mice was significantly higher (P = 0.0145) than that measured in infected WT mice that were not primed. This trend was also seen in the IL-10 response, but the increase was not statistically significant. The overall increase in Th2 cytokines in the primed, infected WT mice contributed to decreasing amounts of IFN-{gamma} in the primed mice. In IL-4KO mice, more moderate increases in IL-5 and IL-10 did not influence the expression of IFN-{gamma}.


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  		TABLE 1 Relative amounts of IFN-{gamma} in CneF-stimulated spleen cell cultures taken from primed and 184A-infected WT or IL-4KO mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The need for Th1-type immunity for protection against infection with Cryptococcus neformans has been firmly established (1), and a detrimental role for Th2-type immunity has also been reported (22). However, the results of this investigation showed that IL-4 was needed for efficient initiation (early development) of CMI during primary infection with C. neoformans. C57Bl/6 mice typically display positive DTH responses (7–10 x 10-3 inches footpad swelling) to cryptococcal antigen at 20 d after infection, but IL-4KO mice on the C57Bl/6 background do not have what would be regarded as a positive DTH response (2.5–5 x 10-3 inches footpad swelling) at that time in the infection. Our finding is consistent with previous observations in IL-4KO mice that described the need for some IL-4 to be present for development of a contact hypersensitivity response to the hapten, trinitrophenyl (23). In the latter case, it was suggested that IL-4 derived from mast cells may stimulate further IL-4 secretion by {gamma}{delta}T cells, and that this IL-4 may be necessary to activate endothelial cells thereby allowing the influx of leukocytes into a DTH reaction site. It has also been reported that small amounts of IL-4 are needed for passive transfer of the contact hypersensitivity response to naïve mice by T cell lines (24). Two reports suggest that IL-4 can have an effect on induction of Th1 responses by its ability to promote bioactive IL-12 from dendritic cells (25) and its synergistic effects with IL-18 that increase IFN-{gamma} production by dendritic cells (26). A role for IL-4 in protection during primary infection has been reported previously for other infections requiring Th1 immunity for protection (14, 15). Detection of high IFN-{gamma} levels at the same time that DTH responses were low during primary infection suggests that the IL-4 deficiency during primary infection could alter trafficking of cells to DTH reaction sites or to infected tissues, and therefore that DTH reactivity is independent of the amount of IFN-{gamma} made by sensitized T lymphocytes present in the peripheral lymphoid tissues.

The effects of IL-4 on survival after a pulmonary infection with C. neoformans were studied using the highly virulent isolate NU-2 because the weakly virulent isolate kills very few mice when given by this route. For a primary, pulmonary infection with NU-2, the lack of IL-4 tended to reduce the survival times but the reduction was significant in only one out of three experiments. Certainly, there was no evidence in these experiments that IL-4 contributed to the poor outcome that is associated with NU-2 infection, as IL-4KO mice did not survive longer than WT mice. These results are in contrast to those reported by Decken and coworkers (27), who found that IL-4–deficient mice infected intravenously with a serotype D cryptococcal isolate survived longer than did infected WT mice. The difference between our results and those of Decken and coworkers may be attributed to differences in route of infection, mouse strain used ([C57Bl/6 X129/Sv/Ev]F2), and serotype of the C. neoformans cryptococcal isolate (serotype D). We have no indication of the relative virulence of the isolate used by Decken and colleagues (27) as compared with our NU-2 isolate, which is more virulent than any other cryptococcal isolate that we have studied (Blackstock and Murphy, unpublished observations), including isolate H99, which is considered to be highly virulent by other investigators (28). On the other hand, our results agree with those of Beenhouwer and associates (29), who reported that IL-4 deficiency did not improve survival of mice infected intravenously with C. neoformans. Different outcomes occur in mice infected with the weakly virulent isolate, 184A, depending upon the route of infection. Approximately 60% of mice survive infection when 184A is given by the pulmonary route (30), whereas 100% of mice die after intravenous infection. For this reason cfu analysis of mice infected by the pulmonary route was used to detect enhanced resistance. This was evidenced by a reduction of cfu in the lungs of IL-4–deficient mice as compared with WT mice. Enhanced resistance in IL-4KO mice was not detected in survival studies after infection by the intravenous route. The failure to improve survival after intravenous infection of IL-4KO mice may be attributed to development of a lower DTH response during primary infection of IL-4KO mice which allowed the organism to have a survival advantage.

Treatment of infected mice with anti–IL-4 enhanced virulence in another model of cryptococcosis (22). In these studies the antibody treatment depleted 26% of the total IL-4 that was detected in the broncoalveolar lavage of infected mice. The fact that IL-4 could be detected in these studies shows that IL-4 production was not completely neutralized in the mice, and the amount of IL-4 detected may have been sufficient to contribute to the development of CMI but insufficient to promote a strong Th2 response. The mouse strain used in the experiments (BALB/c x DBA/2) is one that should genetically favor the development of Th2 responses and antibody production. This difference in infected host used in this study, compared with our model, points to the importance of the genetic characteristics of the host that will influence the type of immunity that is induced during primary cryptococcal infection.

Effects on survival during primary pulmonary infection with the weakly virulent cryptococcal isolate, 184A, could only be studied by examination of cfu isolated from the lungs of IL-4KO and WT mice. In this study we found a significant decrease in the number of cryptococci that were present in the lungs of IL-4KO mice as compared with WT mice on the 20th day after infection but not at time points before Day 20 (data not shown). Even though DTH reactivity was decreased in these mice, there was evidence that enhanced protection was beginning to develop in the knockout mice infected with 184A as reflected in increased cytokine responses by antigen-stimulated spleen cells and decreased cfu in infected lungs under IL-4–deficient conditions. In the cytokine analysis, IL-2 and IFN-{gamma} levels were increased in the IL-4KO mice, whereas IL-5 levels were downregulated and IL-10 levels were not changed from levels in WT mice. During NU-2 infection, Th1 cytokine production by IL-4 KO spleen cells was not significantly different from NU-2–infected WT spleen cells. High IL-5 levels occurred in NU-2–infected WT mice primarily at the 12th day after infection; thereafter, the IL-5 levels were downregulated. These findings point to essential differences in regulation of protective immunity in mice infected with the weakly virulent and highly virulent cryptococcal isolates. Although IL-4 deficiency promotes the development of the protective Th1 response in mice infected with the weakly virulent cryptococcal isolate, it has no effect on the Th1 response in mice infected with the highly virulent NU-2. Previous publications have shown that the highly virulent isolate secretes much more capsular polysaccharide into the serum of infected animals than does the weakly virulent 184A (17). This capsular material is known to downregulate CMI by several different IL-4–independent mechanisms (1, 2, 31).

Splenic cytokine responses to CneF in NU-2–infected C57Bl/6 background mice were routinely lost or very low by the 24th day of infection whether the mice were able to synthesize IL-4 or not. This latter finding is in keeping with our previous report that showed NU-2–infected CBA/J mice become completely unresponsive after the 18th to 20th day of infection, whereas mice infected with 184A continue to exhibit increased Th1 cytokine responses and increased DTH reactions in response to their infection over the first 30 d of infection (17). The finding that NU-2–infected IL-4KO mice also enter an unresponsive phase shows that the unresponsiveness that develops during an NU-2 infection cannot be attributed to the emergence of a strong Th2 response. Consequently, other mechanisms, such as induction of anergy and/or other downregulatory immune responses known to occur in cryptococcosis (31) must be considered as the cause of the unresponsiveness during a NU-2 infection.

The regulatory effects of IL-4 were readily seen in our investigation of immunity to cryptococcal antigen following priming with soluble cryptococcal antigen. After a priming with a small dose of soluble cryptococcal antigen, C. neoformans–infected IL-4KO mice had significantly increased DTH responses and enhanced survival after an intravenous challenge with 184A. These results suggest that compensatory mechanism(s) were available for development and/or expression of the secondary DTH reactions in the primed IL-4KO mice. This mechanism could have been supplied by other cytokines that were available in high levels in the primed mice. Analysis of cytokine production by antigen-stimulated spleen cells of primed and 184A-infected mice revealed that the priming event caused IL-4KO mice to produce more IFN-{gamma} and less Th2 cytokines, especially IL-5, than did WT mice. Thus it appears that the presence of IL-4 during priming significantly changed the protential of splenic cells to produce Th1 and Th2 cytokines when restimulated with CneF. This profile of significantly reduced IFN-{gamma} and increased IL-5 would be expected to effectively reduce protection in WT mice and lower DTH responses to CneF, and that was precisely what we observed in WT mice infected intravenously with the weakly virulent 184A cryptococcal isolate. On the other hand, moderate increases in IL-5 and IL-10 levels in the IL-4KO mice were not sufficient to influence the development of the IFN-{gamma} response and thus the mice did develop a protective DTH response and resisted challenge with 184A.

We have consistently been unable to detect IL-4 in supernatant fluids derived from spleens of infected mice following stimulation in vitro with CneF antigen. This was true in this investigation where C57BL/6J mice were used (data not shown) and in our previous studies on CBA/J mice (17). The inability to detect IL-4 in lymph nodes of C57Bl/6 mice and C.B-17 mice was also reported by Hoag and coworkers (32). However, a number of investigators have reported detection of IL-4 in lung homogenates, broncoalveolar lavage fluids, or antigen-stimulated lung cells that were derived from C. neoformans–infected mice (33, 34). The primary difference in these studies is the site where IL-4 was detected. Uniformly, IL-4 was found in the lung and not in antigen-stimulated lymphocyte cultures derived from peripheral lymphoid tissues of mice that were infected with C. neoformans. This was true whether the draining lymph nodes (32) or spleen cells (17) were studied. These findings suggest that IL-4–producing cells are present in very small numbers in the secondary lymphoid tissues of C. neoformans–infected mice and that they may be concentrated via their migration to the infected lung. An alternative explanation suggests that IL-4 levels in the lung are due wholly, or in part, to other cell types that are known to secrete IL-4. Candidates for this effect would include mast cells, NKT cells, and/or {gamma}{delta}T cells (35). All of these cell types are known to contribute to the development of CMI in various experimental models (24). However, among these cells, mast cells are most able to secrete the highest concentrations of IL-4. In our studies, we elected to study the systemic response to cryptococcal infection because cryptococcosis is a systemic disease. The immune cells that migrate into the lungs originate in the peripheral lymphoid tissues, and therefore it is reasonable to use the systemic response as an indicator of the ability of animals to resist infection.

In summary, this investigation revealed that the effects of IL-4 in cryptococcal disease depends upon the virulence of the cryptococcal isolate used for infection and whether the mice have been primed before infection. Decreased DTH reactions (Th1 immunity) during primary infection with either cryptococcal isolate occurred in the absence of IL-4. On the other hand, exacerbation of infection after priming with cryptococcal antigen was detected in IL-4–sufficient mice infected with a weakly virulent cryptococcal isolate. The role that IL-4 plays during priming suggests that caution in the development of vaccines for C. neoformans is needed to avoid priming the Th2 response that could result in negative consequences to immunized patients who subsequently develop cryptococcosis. Our data suggest that effective cryptococcal vaccines should prime the Th1 response and eliminate Th2 priming.


   Acknowledgments
 
This work was supported by Public Health Service grant HL-59852. The authors are indebted to Anny Alsup, Carrie Tudor, Fredda Schafer, and Donna Thompson for their excellent technical assistance.

Received in original form May 1, 2003

Received in final form June 19, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Murphy, J. W. 1996. Cell-mediated immunity. In The Mycota: Volume VII. Animal and Human Relationships. J. D. Miller and D. H. Howard, editors. Springer-Verlag, New York. 67–97.
  2. Casadevall, A., and J. R. Perfect. 1998. Cryptococcus neoformans. ASM Press, Washington, D.C.
  3. Buchanan, K. L., and J. W. Murphy. 1993. Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. Infect. Immun. 61:2854–2865.[Abstract/Free Full Text]
  4. Huffnagle, G. B., G. B. Toews, M. D. Burdick, M. B. Boyd, K. S. McAllister, R. A. McDonald, S. A. Kunkle, and R. M. Strieter. 1996. Afferent phase production of TNF-{alpha} is required for the development of protective T cell immunity to Cryptococcus neoformans. J. Immunol. 157:4529–4536.[Abstract]
  5. Kawakami, K., Y. Koguchi, M. H. Qureshi, A. Miyazato, S. Yara, Y. I. Y. Kinjo, K. Takeda, S. Akira, M. Kurimoto, and A. Saito. 2000. IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-{gamma} production by NK cells. J. Immunol. 165:941–947.[Abstract/Free Full Text]
  6. Murphy, J. W. 1993. Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response. Infect. Immun. 61:4750–4759.[Abstract/Free Full Text]
  7. Kawakami, K., M. Tohyama, Q. Zie, and A. Saito. 1996. IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans. Clin. Exp. Immunol. 104:208–214.[CrossRef][Medline]
  8. Coffman, R. L., K. Varkila, P. Scott, and R. Chatelain. 1991. Role of cytokines in the differentiation of CD4+ T-Cell subsets In vivo. Immunol. Rev. 123:189–207.[Medline]
  9. Germann, T., M. K. Gately, D. S. Schoenhaut, M. Lohoff, F. Mattner, S. Fischer, S. C. Jin, E. Schmitt, and E. Rude. 1993. Interleukin-12/T-cell stimulating factor, a cytokine with multiple effects on T-Helper type-1 (Th1) but not on Th2 cells. Eur. J. Immunol. 23:1762–1770.[Medline]
  10. Mosmann, T. R., J. H. Schumacher, N. F. Street, R. Budd, A. Ogarra, T. A. T. Fong, M. W. Bond, K. W. M. Moore, A. Sher, and D. F. Fiorentino. 1991. Diversity of cytokine synthesis and function of mouse CD4+ T-Cells. Immunol. Rev. 123:209–229.[Medline]
  11. Wakil, A. E., Z.-E. Wang, J. C. Ryan, D. J. Fowell, and R. M. Locksley. 1998. Interferon {gamma} derived from CD4+ T cells is sufficient to mediate T helper cell type 1 development. J. Exp. Med. 188:1651–1656.[Abstract/Free Full Text]
  12. Cherwinski, H. M., J. H. Schumacher, K. D. Brown, and T. R. Mosemann. 1987. Two types of mouse helper T cell clones III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. J. Exp. Med. 166:1229–1244.[Abstract/Free Full Text]
  13. Fiorentino, D. F., A. Zlotnik, P. Vieira, T. R. Mosmann, M. Howard, K. W. Moore, and A. Ogarra. 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444–3451.[Abstract]
  14. Mencacci, A., G. Del Sero, E. Cenci, C. Fe d'Ostiani, A. Bacci, C. Montagnoli, M. Kopf, and L. Romani. 1998. Endogenous interleukin 4 is required for development of protective CD4+ T helper type 1 cell responses to Candida albicans. J. Exp. Med. 187:307–317.[Abstract/Free Full Text]
  15. Roberts, D. W., D. J. Ferguson, H. Jebbari, A. Satoskar, H. Bluethmann, and J. Alexander. 1996. Different roles for interleukin-4 during the course of Toxoplasma gondii infection. Infect. Immun. 64:897–904.[Abstract]
  16. Cox, F. E. G., and F. Y. Liew. 1992. T-cell subsets and cytokines in parasitic infections. Immunol. Today 13:445–448.[CrossRef][Medline]
  17. Blackstock, R., K. L. Buchanan, A. M. Adesina, and J. W. Murphy. 1999. Differential regulation of immune responses by highly and weakly virulent Cryptococcus neoformans isolates. Infect. Immun. 67:3601–3609.[Abstract/Free Full Text]
  18. Nichols, K. L., S. K. Bauman, F. B. Schafer, and J. W. Murphy. 2002. Differences in components at delayed-type hypersensitivity reaction sites in mice immunized with either a protective or nonprotective immunogen of Cryptococcus neoformans. Infect. Immun. 70:591–600.[Abstract/Free Full Text]
  19. Blackstock, R., N. McElwee, E. Neller, and J. Shaddix-White. 2000. Regulation of cytokine expression in mice immunized with cryptococcal polysaccharide, a glucuronoxylomannan (GXM), associated with peritoneal antigen-presenting cells (APC): Requirements for GXM, APC activation, and IL-12. Infect. Immun. 68:5146–5153.[Abstract/Free Full Text]
  20. Blackstock, R. 2003. Roles for CD40, B7 and major histocompatibility complex in induction of enhanced immunity by cryptococcal polysaccharide-pulsed antigen-presenting cells. Immunology 108:158–166.[CrossRef][Medline]
  21. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350–356.[CrossRef]
  22. Kawakami, K., M. H. Qureshi, T. Zang, Y. Koguchi, Q. Xie, M. Kurimoto, and A. Saito. 1999. Interleukin-4 weakens host resistance to pulmonary and disseminated cryptococcal infection caused by combined treatment with interferon-{gamma}-inducing cytokines. Cell. Immunol. 197:55–61.[CrossRef][Medline]
  23. Asherson, G. L., F. Dieli, G. Sireci, and A. Salerno. 1996. Role of IL-4 in delayed type hypersensitivity. Clin. Exp. Immunol. 103:1–4.[CrossRef][Medline]
  24. Dieli, F., G. L. Asherson, G. C. Romano, G. Sireci, F. Gervasi, and A. Salerno. 1994. IL-4 is essential for the systemic transfer of delayed hypersensitivity by T cell lines - Role of gamma/delta cells. J. Immunol. 152:2698–2704.[Abstract]
  25. Hochrein, H., M. O'Keefe, T. Luft, S. Vandenabeele, R. J. Grumont, E. Maraskovsky, and K. Shortman. 2000. Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells. J. Exp. Med. 192:823–833.[Abstract/Free Full Text]
  26. Fukao, T., S. Matsuda, and S. Koyasu. 2000. Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-{gamma} production by dendritic cells. J. Immunol. 164:64–71.[Abstract/Free Full Text]
  27. Decken, K., G. Kohler, K. Palmer-Lehmann, A. Wunderlin, F. Mattner, J. Magram, M. K. Gately, and G. Alber. 1998. Interleukin-12 is essential for a protective Th1 response in mice infected with Cryptococcus neoformans. Infect. Immun. 66:4994–5000.[Abstract/Free Full Text]
  28. Keller, S. M., M. A. Viviani, M. C. Esposto, M. Cogliati, and B. L. Wickes. 2003. Molecular and genetic characterization of a serotype A MATa Cryptococcus neoformans isolate. Microbiology 149:131–142.[Abstract/Free Full Text]
  29. Beenhouwer, D. O., S. Shapiro, M. Feldmesser, A. Casadevall, and M. D. Scharf. 2001. Both Th1 and Th2 cytokines affect the ability of monoclonal antibodies to protect mice against Cryptococcus neoformans. Infect. Immun. 69:6445–6455.[Abstract/Free Full Text]
  30. Blackstock, R., and J. W. Murphy. 1997. Secretion of the C3 component of complement by peritoneal cells cultured with encapsulated Cryptococcus neoformans. Infect. Immun. 65:4114–4121.[Abstract]
  31. Murphy, J. W., R. L. Mosley, and J. W. Moorhead. 1983. Regulation of cell-mediated immunity in cryptococcosis: II. Characterization of first-order T suppressor cells (Ts1) and induction of second-order suppressor cell. J. Immunol. 130:2876–2881.[Abstract]
  32. Hoag, K. A., N. E. Street, G. B. Huffnagle, and M. F. Lipscomb. 1995. Early cytokine production in pulmonary Cryptococcus neoformans infections distinguishes susceptible and resistant mice. Am. J. Respir. Cell Mol. Biol. 13:487–495.[Abstract]
  33. Abe, K., J. Kadota, Y. Ishimatsu, K. Tomono, K. Kawakami, and S. Kohno. 2000. Th1-Th2 ctyokine kinetics in the bronchoalveolar lavage fluid of mice infected with Cryptococcus neoformans of different virulences. Microbiol. Immunol. 44:849–855.[Medline]
  34. Kawakami, K., M. Tohyama, X. Qifeng, and A. Saito. 1997. Expression of cytokines and inducible nitric oxide synthase mRNA in the lungs of mice infected with Cryptococcus neoformans: Effects of interleukin-12. Infect. Immun. 65:1307–1312.[Abstract]
  35. Weiss, D. L. 2001. Regulation of IL-4 production in mast cells: a paradigm for cell-type-specific gene expression. Immunol. Rev. 179:35–47.[CrossRef][Medline]



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