INTRODUCTION

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We have been investigating Candida albicans mannoprotein (MAN)-specific immunomodulation in a murine model of candidiasis. Injection of MAN intravenously (i.v.) into naive or previously immunized mice stimulates the development of a CD8⁺ effector cell which downregulates MAN-specific delayed hypersensitivity (DH) (24). The CD8⁺ cell can be detected directly in immunized mice treated with MAN, or its presence in splenocyte suspensions can be demonstrated by transfer from MAN-treated mice into immunized mice just prior to footpad testing for DH (18, 24). Cells transferred 2 to 4 days following treatment of donor mice with MAN effectively downregulate DH in immunized recipients, whereas cells transferred prior to 48 h do not. Aside from knowing that CD4⁺ and I-A⁺ cells are required for the production of CD8⁺ effector cells during the first 30 h following the injection of MAN (39), little is known of the process by which the CD8⁺ cells are induced. It is assumed, however, that cytokines play a role.

The specific cytokines, and in what sequence they might function, in the induction of downregulatory effector cells has not been well defined. However, about 10 years ago, Mosmann et al. (47, 48) described the existence of two subtypes of murine CD4⁺ cells, Th1 and Th2, which could be distinguished by the profile of cytokines that they secreted when activated. Numerous investigators have been analyzing the potential roles of Th1 or Th2 cytokines in various immunologic phenomena since that time. Th1 cytokines, interleukin-2 (IL-2) and gamma interferon (IFN-), for example, appear to have prominent roles in cellular immunity, whereas the Th2 cytokines IL-4, IL-6, and IL-10 drive antibody production. Another cytokine, produced predominantly by antigen-presenting cells, IL-12, is believed to be the initiator of cellular immunity (62) and a key modulator of the immune system in general (65, 70). It has been suggested that IL-12 stimulates Th1 cells (62) and simultaneously blocks the differentiation of Th2 cells (45).

Only a few investigators have examined the role of cytokines with respect to downregulation. Notably, Schmitt et al. (61), Ullrich (67), and Rivas and Ullrich (52, 53), working with a model involving the induction of suppression by UV radiation, have determined that UV-induced immune suppression resulted from the secretion of keratinocyte-derived IL-10. IL-4 may also be involved in the immune suppression, as the administration of anti-IL-4 or anti-IL-10 resulted in the abrogation of suppression (53). The administration of exogenous IL-12 prevented the induction of immune suppression by UV and also prevented the activity of preformed suppressor cells (61). In one of the few fungal models in which cytokine involvement in downregulation has been studied, increased secretion of IL-5 and decreased secretion of IFN- and IL-2 were detected (7).

In this study, we analyzed the pattern and kinetics of cytokine mRNA expression in unfractionated spleen cells taken from control and MAN-treated mice. Emphasis was placed on selected cytokines produced by Th1 and Th2 cells, IL-2/IFN- and IL-4/IL-10, respectively, as well as on IL-12. In addition, we measured IL-12p40 and IL-12p70 production by enzyme-linked immunosorbent assay (ELISA). Further, the effect of anti-IL-4 administered to immunized and/or downregulated mice was determined. It was clear that IL-4 participated in the induction of downregulation, but there appeared to be other factors involved as well, as only partial abrogation of downregulatory activity was observed. Moreover, increased serum levels of IL-12p40, a potential antagonist of IL-12p70 (29, 33, 44), may well have permitted the establishment of the CD8⁺ effector cells.

	MATERIALS AND METHODS

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Experimental animals. Male CBA/J mice, 6 to 8 weeks of age, were obtained from Jackson Laboratory, Bar Harbor, Maine. All mice were housed in bioclean hoods and fed mouse chow and water ad libitum.

Preparation and administration of MAN. MAN was prepared as previously described (18) from C. albicans 20A, a serotype A isolate originally obtained from Errol Reiss, Centers for Disease Control and Prevention, Atlanta, Ga., using the method of Peat et al. (51) as modified by Kocourek and Ballou (37). Briefly, the procedure involves autoclaving C. albicans cells in a citrate buffer to solubilize the mannoprotein, precipitation of the polysaccharide with Fehling's solution, dissolution of the polysaccharide-copper complexes in HCl to remove the copper, and then dialyzing the preparation against distilled water to remove any low-molecular-weight components. MAN extracted in this manner contains approximately 5% protein, and mannose is the only sugar detectable (18, 51). The extract was lyophilized and stored in a desiccator prior to use.

Preparation of splenic homogenates and isolation of poly(A)⁺ RNA. Total RNA was first isolated by homogenizing spleens in 2 ml of guanidinium buffer (4 M guanidinium isothiocyanate, 0.5% sarcosine, 25 mM sodium citrate, 0.7% -mercaptoethanol). This isolation procedure is based on the guanidinium thiocyanate-phenol-chloroform extraction method described by Chomczynski and Sacchi (12). We modified it to include a second extraction. After centrifugation at 13,000 × g for 15 min, the RNA in the aqueous phase was precipitated by isopropanol. RNA precipitates were dissolved in 1 ml of TE buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA [pH 7.5]) and extracted a second time. The precipitates were then washed once in 75% ethanol and resuspended in 200 µl of 0.2% diethyl pyrocarbonate in water. Poly(A)⁺ RNA was isolated by first heating a mixture containing 250 µg of RNA in 450 µl of a solution containing 360 µl of 0.1% diethyl pyrocarbonate water, 75 µl of binding buffer (60 mM Tris-HCl, 6 mM EDTA, 3 M NaCl [pH 7.5]), and 15 µl of polystyrene latex Oligotex-dT beads (Qiagen, Chatsworth, Calif.) to 65°C for 5 min. Thereafter, the suspension was incubated at room temperature for 30 min, and the Oligotex-dT beads were washed in TE buffer. Poly(A)⁺ RNA was eluted at 80°C from the polystyrene latex-oligo(dT) beads with elution buffer (5 mM Tris [pH 7.5]). Poly(A)⁺ RNA was quantitated for each sample by using multiple dilutions applied to DNA Dipsticks (Invitrogen).

Reverse transcriptase-mediated PCR (RT-PCR) for detection of cytokine mRNA. To determine whether a particular cytokine mRNA was expressed, 300 ng of poly(A)⁺ RNA, isolated as described above from murine spleens stimulated with MAN, was reverse transcribed in the buffer supplied by the manufacturer in the presence of random hexamer with 200 to 400 U of SuperScript II RNase H reverse transcriptase or Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, Md.). The reaction was carried out at room temperature for 30 min, followed by 90 min at 37°C. The reverse transcriptase was inactivated at 95°C for 5 min, and the cDNA was precipitated in ethanol. After the precipitate was washed in 75% ethanol, 15% of the reverse-transcribed RNA, the equivalent of 40 ng of input RNA, was used in PCR for the detection of each cytokine gene in question or for the detection of the housekeeping gene G3PDH (glyceraldehyde-3-phosphate dehydrogenase). The amplification reaction included combining the cDNA with 1.25 U of Taq polymerase, 200 µM deoxynucleoside triphosphates, 1 µg of each primer, 2 to 3 mM MgCl₂, and PCR buffer (Gibco BRL). Positive (concanavalin A-stimulated splenocyte extracts) and negative (water) controls were included in each assay. Reactions were assembled with positive-displacement pipettes and brought to 65°C prior to the addition of Taq polymerase. Each 50-µl sample was overlaid with 75 µl of mineral oil (Sigma Chemical Co., St. Louis, Mo.) and placed in a thermal cycler (Robocycler 40 [Stratagene, La Jolla, Calif.] or Apolitron I [Thermolyne, Dubuque, Iowa]) with temperatures of 95°C for denaturation, 58°C for annealing, and 72°C for extension, with the first 3 of 35 total cycles having extended denaturation and annealing times. Various numbers of cycles for each cytokine were compared in early experiments, and 35 was in the linear range for all cytokines tested. Primers were synthesized by the Midland Certified Reagent Company (Midland, Tex.) according to sequences selected by one of the authors. The sequences of 5' sense primers and 3' antisense primers used in this study and their amplified fragment DNA sizes are summarized in Table 1.

Quantitation of IL-12p40 mRNA. Initially the relative quantities of all cytokines were determined visually from photographs of the gels. All poly(A)⁺ samples from a given experiment representing each of the time intervals tested were subjected to RT-PCR for a specific cytokine mRNA at the same time, using the same percentage of input cDNA; i.e., each reaction was begun transcribing 300 ng of mRNA, and 15% of each transcribed mixture was subjected to PCR and then electrophoresed in the same gel or in two gels placed in the same chamber at the same time. Photographs of the gels were then evaluated independently by three individuals, and the intensity of bands was rated on a scale of 0 to +3. The mean ± standard error of the mean could then be determined by combining the data from all animals. IL-12 was selected for more accurate quantification by quantitative competitive RT-PCR (QC-RT-PCR). Specifically, the amount of IL-12p40 mRNA expressed in a particular sample was determined by the method of Bost and Clements (6) except that the actual quantification was performed by densitometry rather than radiolabeling. Competitor DNA, similar to but demonstrably different in size from IL-12p40 cDNA, but which could be amplified by the same pair of primers, was produced by RT-PCR amplification of plasmid DNA.

Quantitation of secreted IL-12p40 and IL-12p70. Secretion of IL-12p40 into sera was quantified by a capture ELISA as described previously (35). The monoclonal antibody C15.6, which recognizes monomeric or dimeric IL-12p40, was used to coat microtiter plates, and the IL-12p40 captured by this monoclonal antibody was detected by using the biotinylated monoclonal antibody with specificity for a different determinant of IL-12p40, C17.8. A hybridoma secreting the monoclonal antibody C15.6 was kindly provided by G. Trinchieri (Wistar Institute, Philadelphia, Pa.), and the biotinylated antibody produced by C17.8 was purchased from PharMingen (San Diego, Calif.). The sensitivity of this assay was determined to be approximately 10 pg of IL-12p40 per ml.

Preparation of monoclonal anti-IL-4. Anti-IL-4 antibody was harvested from ascites following intraperitoneal (i.p.) injection of 5 × 10⁶ cultured 11B11 hybridoma cells (American Type Culture Collection, Rockville, Md.) into nude mice 10 days after i.p. injection of 0.5 ml pristane (Sigma). The induced ascites were collected beginning at day 10, and purified immunoglobulins were extracted by using a protein A affinity column (Bio-Rad Chemical Division, Richmond, Calif.). The procedures outlined by the manufacturer of the column were followed. Protein was estimated in the purified immunoglobulin preparations by absorbance readings at 280 nm, and mice were treated with the antibodies, based on micrograms of protein per milliliter. Each mouse received 200 to 400 µg of protein/dose i.p. These doses were selected empirically, using as a guideline doses used successfully by others (42, 54, 59).

Immunization of mice and measurement of MAN-specific DH. Mice were immunized by the cutaneous inoculation of 10⁶ viable C. albicans on two occasions 2 weeks apart as described previously (28). Animals immunized in this manner develop maximum levels of DH 1 week following the second candidal challenge (19). DH was measured as previously described following the injection of 20 µl of NPS containing 30 µg of MAN into the footpads of mice (18). The footpads were premeasured with calipers and then measured again at 15 min and at 4 and 24 h after the injection of antigen. Since DH in the mouse peaks 24 h after the injection of antigen (reviewed in reference 14), data are reported for that time point.

Statistics. Group sizes for in vivo studies of DH ranged from 6 to 8. The data were analyzed by using the nonparametric Mann-Whitney test.

	RESULTS

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Cytokine mRNA expressed by splenocytes in mice stimulated with MAN. We had shown previously that the injection of MAN i.v. into mice stimulated the production of splenic CD8⁺ effector cells, which downregulated DH when transferred to immunized mice (24). Functional CD8⁺ cells were not present in transfer suspensions until 2 to 4 days after the injection of MAN (24), and CD4⁺ and I-A⁺ cells were required during the first 30 h after the injection of MAN for the induction of the CD8⁺ cells (39). Therefore, to obtain evidence of cytokine involvement during the induction of this regulatory phenomenon, splenocytes were tested for cytokine mRNA at selected intervals beginning at 2 h and continuing through 96 h after the introduction of MAN. As the experiments summarized below and in Fig. 1 were viewed as surveys designed to identify major changes in cytokine synthesis over time, and since a densitometer was not available to us at the time these analyses were done, it was felt that crude visual examinations would provide the data required for more in-depth studies to follow.

View larger version (79K): [in this window] [in a new window]   FIG. 1.   Composite photograph of RT-PCR products detected with cytokine or housekeeping gene-specific probes, using splenocytes of mice taken at various intervals following the injection of saline or 500 µg of C. albicans mannan i.v. P, positive control, splenocytes stimulated with concanavalin A; N, negative control. The negative control lane and the molecular weight marker lane are reversed on the IFN- strip. Molecular weight markers were placed at each side of the gel.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Quantitation, using QC-RT-PCR and a competitive inhibitor of IL-12p40, in splenocytes taken from mice at selected intervals following the i.v. injection of saline, represented on the figure as 0 h, or C. albicans mannan. The saline-injected animals were sacrificed along with the animals inoculated with MAN 2 h prior to sacrifice. The insert is a representative gel of the assay and shows the gel from which the 2-h MAN determination was made. The concentrations of competitor shown range from a high of 7,500 pg to a low of 10 pg.

Partial abrogation of downregulatory activity in vivo by administration of anti-IL-4 to immunized and MAN-treated mice. IL-4 was selected for additional study for the following reasons: (i) it was the cytokine for which the greatest increase in the number of spot-forming cells (SFC) was noted in MAN-treated mice during the induction of CD8⁺ effector cells (40), and (ii) IL-4 has been described as a cytokine required for the induction and elicitation of contact sensitivity (CS) (2, 60, 69). Therefore, if IL-4 is a critical cytokine for the expression of DH to MAN, it would be unlikely to play a role in downregulation, as suggested by the ELISPOT data.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   DH in control and immunized mice, some of which were treated during the induction and/or expression phases with NPS, MAN, and/or monoclonal anti-IL-4 antibody or rat serum immunoglobulin (Table 4). In the experiment shown, animals treated during both the induction and expression phases received a total of 2 mg of protein of the anti-IL-4 preparation or rat immunoglobulin, those treated during only the induction phase received 1,200 µg of protein, and those treated during the expression phase received 700 µg of protein. NS, not significant. P values were determined by the Mann-Whitney test.

	DISCUSSION

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The most interesting observations made here over the 96-h observation period following the administration of MAN include (i) a reduction in mRNA synthesis for IL-12p40 within the first 2 h and continuing through 24 h, (ii) secretion of IL-12p40, beginning with levels barely detectable at 12 h, peaking at 48 h, and returning to barely detectable at 96 h, (iii) complete lack of secretion of IL-12p70 at any time after the administration of MAN, (iv) little or no apparent synthesis of mRNA specific for IFN- and IL-4 for the first 48 h but obvious production thereafter, and (v) synthesis of IL-10 mRNA throughout the entire period. In addition, partial abrogation of downregulatory activity was affected by the administration of anti-IL-4 to immunized animals during the induction phase of the downregulatory activity.

IL-12 is a 70-kDa heterodimeric cytokine composed of covalently linked 35-kDa (p35) and 40-kDa (p40) subunits whose activity was first described by Kobayashi et al. (36). It is produced predominantly by macrophages and B cells (10, 15), and it is generally believed to link the innate and acquired immune systems (reviewed in reference 65). IL-12 induces the production of several cytokines, including IFN- and IL-2 (66, 70), from T lymphocytes and natural killer (NK) cells in vitro as well as in vivo (26). Moreover, IL-12 induces the differentiation of Th1 cells from naive T cells (Th0) (34), thus initiating cell-mediated immunity. It regulates the proliferation of not only Th1 cells (34) but also cytotoxic T lymphocytes (66). This cytokine, therefore, represents an important regulatory bridge between innate resistance and adoptive immunity (65).

Since only IL-12p40, and not IL-12p70, the former of which is a known antagonist of IL-12 bioactivity (29, 33, 44), was secreted following the introduction of MAN into naive animals, there was never the opportunity for DH to be initiated. However, the phenomenon observed here is not simply a lack of initiation of DH, in that MAN-specific downregulatory cells that can function to inhibit an established DH response are also produced (24). Thus, it is likely that other cytokines, for example, IL-4 and/or IL-10, play a role during the inductive phase. While we have no evidence at this time with regard to IL-10, we have shown that IL-4-secreting cells were increased in elevated numbers beginning at about 24 h following the introduction of MAN (40) and that the administration of anti-IL-4 to immunized MAN-treated mice partially abrogated the downregulatory activity. Moreover, even if some bioactive IL-12 was produced but remained undetected in the MAN-treated animals, it has been reported in other systems that IL-4 takes precedence over IL-12 when the two cytokines are present at the same time (34, 64, 66).

IL-10 is as interesting as IL-12 with regard to regulation of cell-mediated immunity, however. IL-10 was originally described as a factor which prevented the proliferation of Th1 cell clones by inhibiting IFN- and IL-2 production (21, 22). Subsequently, Fiorentino et al. (23) demonstrated that IL-10 acted on antigen-presenting cells to inhibit cytokine synthesis. IL-10, in fact, has been shown to have many different functions (reviewed in reference 46), some of which are stimulatory (11, 41) and some of which are inhibitory (17, 20, 38, 67, 71). The precise role of IL-10 in this MAN-specific system is unknown at this time, since we have not yet had an opportunity to investigate the secretion of IL-10 after MAN administration or the effects of ablation of IL-10 activity in vivo.

The data presented here, as well as those in another study from our laboratory (40), are not consistent with the hypothesis that IL-4 is a critical cytokine in the initiation and elicitation of DH to MAN, such as has been proposed for CS to chemicals. CS is considered one manifestation of cellular immunity, specifically DH, and two groups of investigators have proposed that IL-4 is critical for both its induction (60) and elicitation (69). There are conflicting data (27), however. Weigmann et al. (69) speculated that these seemingly contradictory activities of IL-4 could be explained on the basis of concentration of IL-4; higher concentrations were inhibitory and lower concentrations were enhancing. The data obtained, therefore, would depend on the efficiency with which IL-4 was removed or inhibited in vivo. We have collected no additional data at this time that would allow us to state unequivocally how IL-4 and other factors contribute to the induction of MAN-specific downregulation in murine candidiasis. Reports of inhibition of cell-mediated immunity and reactive nitrogen oxide induced by IL-4, IL-10, and transforming growth factor  in parasitic infections (63), along with data showing the inhibition of both candidacidal activity and nitric oxide production in IFN--activated macrophages by IL-4 and/or IL-10 (9), may provide clues for future investigations of the mechanism of action of cytokines in MAN-induced suppression.

Several laboratories have been investigating additional relationships between various cytokines and C. albicans (55-57) or C. albicans MAN (3). IL-12 production has been shown to correlate with the induction of Th1 type responses in murine candidiasis, and the neutralization of IL-12 by administration of anti-IL-12 antibody abrogates resistance to C. albicans (55-57). Using human cells in vitro, Ausiello et al. (3) determined that mannoprotein did not consistently stimulate the expression of IL-1, IL-5, and IL-10 but did induce long-lasting production of mRNAs for IL-1, tumor necrosis factor alpha, and IL-6, along with appreciable levels of granulocyte-macrophage colony-stimulating factor, IFN-, and IL-2. The stimulation of human cells in vitro appears to be quite different from the stimulation of murine cells in vivo, particularly with respect to the stimulation of IFN- and IL-2, as evidenced from both the mRNA studies here and the ELISPOT data presented elsewhere (40).

The inability or reduced ability to respond to a normally immunogenic preparation of an antigen has been a highly controversial area over the past decade, and the validity of the terms suppression and suppressor cell have been debated with considerable fervor (30). Despite the debate, there is no question that the phenomenon occurs; it has been demonstrated in fungal models by us (8, 18) and others (5, 13, 16, 32, 49), in bacterial and parasitic systems (reviewed in reference 68), and in systems involving CS (reviewed in reference 1). There has been something of a renewed interest in suppressive phenomena; IL-10 production by autoreactive CD8⁺ T-cell clones has been described (31), and IL-10 epitopes (43) have been associated with antigen-specific T suppressor cells. Moreover, Nanda et al. (50) have described a unique pattern of lymphokine synthesis involving the cosynthesis of IL-10 and IFN- in selected antigen-specific suppressor T-cell clones. IFN- message was not upregulated during the induction phase of suppression in the MAN-specific model, i.e., 48 h after the injection of MAN, but it was thereafter. Finally, Buchanan and Murphy (7) reported increased production of IL-5 in suppressed mice, with decreased production of IFN- and IL-2. IL-4 was unchanged.

The role of these downregulatory cells in protective immunity in experimental candidiasis has been incompletely explored. It has been shown that when downregulatory cells are induced in immunized animals, they have no effect on protective immunity when animals are challenged i.v. (25). Since there appears to be a dichotomy of protective mechanisms against C. albicans, however, with cellular immunity being critical against mucosal disease (4), downregulatory cells may well modulate protective immunity at that level. Investigations of immunoregulatory cell involvement in protection at the mucosal level await the development of a suitable animal model in which to demonstrate it.

In summary, our data are suggestive of a role for IL-4, acting in the absence of bioactive IL-12 and presence of potentially antagonistic IL-12p40, in the induction and maintenance of MAN-specific T-cell suppression. Perhaps IL-10 contributes to the induction of the CD8⁺ effector cells as well, but since only message, and not secretion, was measured for that cytokine, there are no data to support that hypothesis. As a next step, however, it would be appropriate to determine whether in vivo treatment with IL-12 or with anti-IL-10 prevents the induction of the MAN-specific immunomodulatory cells. The most definite experiments will likely involve knockout mice.