INTRODUCTION

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Chronic disseminated candidiasis (CDC) is a serious infectious complication that occurs in patients suffering prolonged periods of neutropenia (25). Successful treatment of CDC typically requires protracted courses of antifungal therapy (56). Parenchymal lesions within the liver in which yeast and pseudohyphae can be found become apparent during recovery from neutropenia and can progress in size, often with a strong local inflammatory response. This observation suggests that inhibitory factors produced locally in response to fungal infection could impair an effective immune response and consequently, as circulating neutrophils and monocytes recover, contribute to the pathogenesis and prognosis of CDC. Several aspects of the host response to Candida albicans have been defined through studies of humans with CDC and animal models of disseminated infection. A suitable animal model for C. albicans infection of the liver in neutropenic rabbits provides an excellent opportunity to investigate the pathophysiological model and to develop new therapeutic strategies (54, 55).

An effective phagocytic response absolutely depends on a balance between pro-and anti-inflammatory cytokines and on T-helper cells. Studies of animals models of C. albicans infection have illustrated the protective role of T-helper lymphocyte type 1 (Th1) cytokines and the suppressive effect of Th2 cytokines on the host response to infection (34, 42). For example, interleukin-12 (IL-12) is required for Th1 differentiation in murine candidiasis (40), and production of this cytokine by neutrophils correlates with a protective response (41). Exogenous IL-12 is effective in protecting neutropenic hosts susceptible to infection and can enhance the host phagocytic response to C. albicans (41). The role of Th2 cytokines is less clearly defined, but current data suggest that they impair the host response to C. albicans (10, 29, 35, 49). Measurement of high circulating levels of IL-10 in patients with CDC (38) indicates a shift toward the Th2 response. IL-10 is a potent inhibitor of cytokine synthesis in human monocytes (15) and can inhibit the release of proinflammatory cytokines, such as IL-1 and tumor necrosis factor alpha (8, 9). IL-10 inhibits the phagocytic activity of human neutrophils (7) and suppresses antifungal activities of human monocytes against the pseudohyphae and blastoconidida of C. albicans (36).

Transforming growth factor  (TGF-) is another important inhibitory cytokine (5, 27), but the role that it plays during infection with C. albicans is not clearly defined. Release of TGF- down-regulates activated monocytes and macrophages, suppressing gamma interferon (IFN-)-induced production of nitric oxide (51), which would favor the dissemination and progression of C. albicans infection. TGF- is generally thought to influence the differentiation of naive CD4⁺T cells toward the Th2 profile (27), although it has been shown to inhibit Th2 differentiation (21). Murine models characterized by disruptions in the TGF- pathway are notable for a proinflammatory phenotype, with spontaneous differentiation and activation of T cells producing IFN- and IL-4 (20, 26, 46). Studies with mice have suggested a role for TGF- in determining the response to C. albicans (48), by demonstrating that production of TGF- quickly follows infection with a nonvirulent vaccine strain of C. albicans, and have shown that this role might be important in the development of resistance. However, the observation that administration of exogenous TGF- could promote the development of a Th1 response in these mice highlights the complex local effects of this cytokine and suggests that TGF- might play a critical regulatory role in the host response to C. albicans infection (48).

Although these studies have shown that systemic administration of a neutralizing anti-TGF- antibody can affect the course of C. albicans infection in mice, the cellular source and local production of TGF- have not been clearly determined. The objective of this study was to demonstrate that local production of TGF- accompanies the granulomatous response to tissue invasion in CDC and to investigate whether human monocytes challenged by C. albicans produce biologically active TGF-. The results suggest an important role for TGF- in the pathophysiology of CDC.

	MATERIALS AND METHODS

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Human liver biopsy specimens. Seven patients with biopsy-documented CDC were identified, and 5-µm sections were prepared from archival specimens that had previously been fixed in formalin and embedded in paraffin. Specimens were processed for immunohistochemical analysis of TGF- as described below.

Rabbit model of CDC. New Zealand White rabbits (weight, 2.5 to 3.5 kg; Hazleton, Denver, Pa.) were used for all experiments and were given water and standard rabbit feed ad libitum according to National Institutes of Health guidelines (12). The immunosuppressive regimen and supportive care measures were as previously described (54, 55). Briefly, cytosine arabinoside (Upjohn Pharmaceuticals, Kalamazoo, Mich.) was administered intravenously at 440 mg/m² on days 1 through 5 and on days 8 to 9 and days 13 to 14 to produce profound and persistent neutropenia, respectively, and starting on day 4, rabbits received intravenous administration of ceftazidime (Glaxo Pharmaceuticals, Research Triangle Park, N.C.) at 75 mg/kg, twice daily, gentamicin (Baxter Health Care Corp., Deerfield, Ill.) at 5 mg/kg every other day, and vancomycin (Eli Lilly & Co., Indianapolis, Ind.) intravenously at 15 mg/kg daily to prevent the occurrence of invasive bacterial infections during neutropenia. An inoculum of 10³ CFU of C. albicans was administered on day 6 as previously described (22).

Immunohistochemical detection of TGF- isoforms in human and rabbit tissues. Sections 5 µm thick were stained with hematoxylin and eosin for routine histological evaluation. Additional sections were evaluated with isoform-specific anti-TGF- antibodies directed against TGF-1 (18), TGF-2 (16), and TGF-3 (17), followed by peroxidase staining as previously described (18). The sections were also evaluated using a biotinylated mouse monoclonal antibody that was raised against mature, active TGF-2 but can recognize and neutralize the activity of all three isoforms (clone 1D11; Genzyme, Cambridge, Mass., and now available through R&D Systems, Minneapolis, Minn.).

Monocyte infection and preparation of conditioned media. Peripheral blood monocytes were isolated by a two-step procedure, automated leukopheresis followed by counterflow elutriation (model J-6M centrifuge; Beckman Instruments, Fullerton, Calif.) (53). Cell viability was determined to be >95% by trypan blue exclusion. Morphological analysis by using modified Wright-Giemsa stain and nonspecific esterase stain confirmed that >95% of the isolated cells were monocytes. In preparation for ex vivo studies, monocytes were washed twice in RPMI without fetal calf serum and kept on ice throughout preparation. The monocytes were resuspended in RPMI at 10⁶/ml in a total volume of 30 ml, immediately placed in a 5% CO₂ water incubator, and subjected to either C. albicans strain 86-21 challenge or a sterile-water control treatment. The C. albicans strain is a clinical specimen and has been previously studied (37). Monocyte suspensions were challenged with C. albicans at a multiplicity of infection (MOI [target-to-effector cells]) of 10:1, 1:1, and 1:10 in duplicate. Supernatants were collected at 8, 12, 24, and 48 h after initial infection. The viability of cells was assessed by trypan blue stain exclusion. For the time points collected, the viability of monocytes exposed to an MOI of 10:1 was less than 50%. For the other conditions, the viability was >95% at the time of collection. Supernatants were collected and frozen at 70°C until analysis.

TGF- bioassay. A modified Mv1Lu bioassay was used (13). Cells were plated at 2 × 10⁵ cells per well in 24-well plates with 1 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and incubated for 8 to 12 h at 37°C to ensure complete adherence. The medium was aspirated and replaced with serial dilutions of each conditioned medium (diluted in Dulbecco's modified Eagle's medium containing 0.2% fetal bovine serum) in the presence or absence of 30 µg of either the panspecific mouse monoclonal anti-TGF- blocking antibody 1D11 or control IgG per ml; additional wells were treated with medium plus TGF- standard concentrations in a total volume of 500 µl/well (each condition was tested in triplicate). The plates were subsequently incubated for an additional 24 h, and 1 mCi of [³H]thymidine was added for the final 2 h of the incubation. The medium was aspirated and replaced with 50 µl of trypsin-EDTA/well, and the cells were incubated for 30 min at 37°C before being harvested onto 24-well filter plates, which were processed with a Top Count microplate scintillation reader as specified by the manufacturer (Packard Instrument Co., Meriden, Conn.).

TGF- SELISA. Culture supernatants were collected as above for determination of total TGF- by the Quantikine SELISA (R&D Systems). Total TGF- was measured after acidification to activate latent TGF- followed by neutralization specified by the manufacturer, with standard curves for TGF- generated with known amounts of purified recombinant human TGF-1.

	RESULTS

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Production of TGF- accompanies the local inflammatory response to C. albicans in vivo. We examined the expression of TGF- within the inflammatory liver granulomas of seven patients with documented CDC. Immunohistochemical studies demonstrated intense staining for TGF-1 within the extracellular matrix surrounding infiltrates of leukocytes within the liver parenchyma (Fig. 1A to D) and revealed an extensive accumulation of TGF- protein encasing necrotic foci that resulted from the intense local inflammatory reaction (Fig. 1E).

View larger version (129K): [in this window] [in a new window]   FIG. 1.   Immunolocalization of TGF-1 within the extracellular matrix of hepatic granulomas of patients with CDC. Tissue sections from seven patients with CDC were examined by immunohistochemistry with a polyclonal rabbit antibody (designated CC) that detects extracellular, matrix-associated TGF-1. Sections stained with normal rabbit serum (A) or the CC antibody (B to E) are shown.

View larger version (159K): [in this window] [in a new window]   FIG. 2.   Immunolocalization of TGF-1 and TGF-3 in hepatocytes adjacent to inflammatory granulomas. Tissue sections from patients with CDC were examined by immunohistochemistry with either normal rabbit serum (A) or polyclonal rabbit antibodies to TGF-3 (B and C), or TGF-1 (D). Results are representative of staining patterns observed in all sections examined.

View larger version (180K): [in this window] [in a new window]   FIG. 3.   Immunolocalization of TGF- in sections of liver taken from neutropenic rabbits infected with C. albicans. (A to D) Sections were preblocked with normal goat serum prior to incubation with the rabbit polycolonal CC antibody directed against extracellular TGF-1 (A and C) and compared with control staining with normal rabbit serum (B and D). (E and F) The murine monoclonal panspecific anti-TGF- antibody 1D11 was biotinylated and used to detect intracellular, active TGF- within hepatocytes of C. albicans-infected livers (E) and compared to staining with normal mouse serum (F).

Monocyte infection by C. albicans results in the secretion of biologically active TGF-. Infiltrating leukocytes might also contribute to the local production of TGF- in the liver following infection with C. albicans. To study this possibility, monocytes were isolated by leukapheresis and counterflow elutriation and cultured at specified ratios of blastoconidia to monocytes in serum-free medium. Culture supernatants were then collected at time intervals ranging from 8 to 48 h, and the presence of biologically active TGF- was determined by the ability to inhibit growth of a TGF--sensitive mink lung epithelial cell line in a manner reversible with a neutralizing. TGF--specific antibody. Supernatants from cultures of 1.5 × 10⁶/ml monocytes at a blastoconidia-to-monocyte ratio of 1:1 contained more than 1 ng of biologically active TGF- per ml. As shown in Fig. 4, substantial amounts of TGF- could be detected as early as 12 h, and similar results were obtained in assays of supernatants collected at later time points. A determination of total TGF- was performed on the same culture supernatants following a transient acidification to release any mature TGF- remaining associated with the latency-associated protein. The results shown in Fig. 5 directly correlate with values determined by the bioassay and suggest that the majority of the TGF- released into the medium by infected monocytes is biologically active.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Production of active TGF- by C. albicans infected human peripheral blood monocytes. Peripheral blood monocytes were collected as described in Materials and Methods and cultured in defined medium with blastoconidia for the indicated time intervals. (A) The medium was collected and assayed for the presence of TGF-, and the result was compared to a standard curve generated by the response to recombinant human TGF-1 (B and C) The conditioned medium samples collected at 12 h (B) and 24 h (C) each contained an activity capable of inhibiting the proliferation of Mv1lu cells (black bars) in a manner reversible by a TGF- neutralizing antibody (white bar) but not by a nonneutralizing, isotype control IgG (hatched bar), thereby demonstrating TGF-- specificity of the neutralization of growth inhibition. Levels of TGF- were most notable at target-to-effector-cell (T:E) (yeast/monocyte) ratios of 10:1 and 1:1. The results in panels B and C are the mean of three separate experiments for conditioned medium at a 1:2 dilution.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Production of active TGF- by C. albicans-infected human peripheral blood monocytes. In addition to a bioassay of conditioned medium, we performed a SELISA for TGF-1 as described in Materials and Methods. The results for acid-activated conditioned medium at a 1:2 dilution directly correlate with the level of activity as determined by the inhibition of Mv1lu cell growth. These results suggest that the majority of the TGF- secreted by Candida-infected monocytes has been processed to a biologically active form. Ratios of yeast to monocytes (T:E) were as indicated and were obtained with the same culture supernatants used for experiments in Fig. 4.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Our results demonstrate that active TGF- is produced by hepatocytes and infiltrating monocytes within inflammatory granulomas during CDC. Furthermore, we show an induction of active TGF- in hepatocytes and an accumulation of extracellular, matrix-associated TGF- in areas surrounding both inflammatory granulomas and the residual necrotic foci that are characteristic of CDC. We present evidence that local production of TGF- is not unique to CDC in humans but that it also occurs during C. albicans infection in immunocompromised rabbits following prolonged periods of neutropenia. Infection of monocytes with C. albicans leads to the secretion of active TGF-, clearly implicating leukocytes as an additional source of this cytokine in CDC. Since clinical expression of CDC is often accelerated during and after recovery from neutropenia and monocytopenia, we infer that the high local concentration of TGF- participates in the suppression of host defenses.

Previously, it has been shown that the immunoregulatory properties of TGF- affect the differentiation and function of nearly every leukocyte subset (27). TGF- primarily inhibits the differentiated function of immune cells (i.e., phagocytes), although several studies suggest that it can also enhance the function of lymphocytes and macrophages. For example, femtomolar concentrations of TGF- are chemotactic for human peripheral blood monocytes and neutrophils, suggesting a critical role in recruitment to sites of injury or inflammation. Mice lacking the MADH3 gene, encoding the TGF- receptor-activated Smad3, have defective chemotactic responses to TGF- in neutrophils, monocytes, and keratinocytes; they spontaneously develop mucosal abscesses with nonpathogenic Providencia spp. (14, 59). TGF- can also contribute to the formation of inflammatory foci by enhancing the expression of several integrin receptors on monocytes, namely, LFA-1, VLA-3, and VLA-5 (3, 52). TGF- enhances phagocytosis by induction of the expression of FcRIII receptors on circulating monocytes (57). These data suggest that local production of TGF- within tissues may promote the infiltration of mononuclear cells responding to pathogens such as C. albicans.

On the other hand, TGF- is also known to inhibit the function of immune cells (i.e., lymphocytes and phagocytes) postactivation. For example, TGF- is a potent inhibitor of the production of reactive oxygen radicals and nitrogen intermediates by cells activated by either IFN- or bacterial lipopolysaccharide. (6, 51). Interestingly these activating signals are also recognized for their ability to induce monocytes and macrophages to release TGF- in an active state, through mechanisms that involve the serine protease plasmin and tissue type II transglutaminase (32, 33). C. albicans infection of IFN- and lipopolysaccharide-activated murine peritoneal macrophages can also suppress the production of nitric oxide, although this effect has been described as being independent of induction by TGF-, primarily because it was not neutralized by a blocking antibody to TGF- (11). While this autocrine activity generally is believed to function as an important feedback-inhibitory mechanism for limiting the extent and duration of an inflammatory response, it is also well recognized as a microbial escape mechanism in human and murine forms of parasitic infection. Indeed, infection of host macrophages with either Leishmania or the protozoan parasite Trypanosoma cruzi results in the secretion of the mature, active TGF- that suppresses microbicidal activity against the parasite and thereby enhances the proliferation of the pathogen (1, 2, 30, 47). The effects of TGF- on the development of host defense pathways have also been studied during infection with Mycobacterium tuberculosis and leprosy. TGF- is produced by M. tuberculosis-infected macrophages (39), and neutralization of TGF- normalizes lymphocyte proliferative responses to the standard purified protein derivative; partially restores blastogenesis to candidal antigen, and increases IFN- production, indicating that TGF- is an important mediator of immunosuppression in tuberculosis (23, 24). In leprosy, two different patterns of TGF- isoform expression are seen in the polar forms of leprosy in skin biopsy specimens; in the paucibacillary form, expression of the latent form of TGF-1 was detected, whereas in the tuberculoid form, high levels of the active isoforms were detected (58). Our data provide preliminary evidence that C. albicans could utilize a comparable strategy in the neutropenic host, similar to the observed response to M. tuberculosis and selected parasitic infections. In our CDC study, the active isoform of TGF-1 is highly expressed and could be a contributing factor to CDC pathogenesis.

Another important component of the host response to C. albicans is the development of an effective Th1 response. The contribution of TGF- to Th cell differentiation is complex, but early studies suggested that TGF- promotes the differentiation of staphylococcal enterotoxin B-stimulated CD4⁺ T cells toward the Th1 phenotype (31). Conversely, others have demonstrated that TGF- inhibits IL-12-induced Th1 development and IFN- production (44, 45). More recent studies involving polyclonal activation of naîve CD4⁺ T cells and ovalbumin-specific Th cells have demonstrated that a mixture of IL-4 and TGF- can promote the development of either Th1 or Th2 cells in a concentration dependent manner (28). However, the induction of a Th1 phenotype was dependent on the presence of IFN-, which has recently been shown to block TGF- receptor-mediated signaling though the induction of the inhibitory Smad7 (50).

The establishment of an effective Th1 response in mice with systemic candidiasis is thought to be dependent on several factors, including the production of IL-12 (43) and the early presence of IL-4, which can prime neutrophils for production of IL-12 (29). Studies with mice have shown that IFN- is required for an effective Th1 response to C. albicans but also suggest that presence of TGF- could favor a Th1 response (48). Taken together, these observations suggest that multiple pathways contribute to the differentiation of naîve T cells and that the intracellular cross talk between pathways, including TGF- and Th1 cytokines (4, 50, 60), could unfavorably tip the balance of the host immune response in the neutropenic host.

The current data collectively demonstrate that TGF- is an important determinant of the host response to systemic candidiasis. As a consequence of the suppressive effects of TGF- on the phagocytic response, the production of active TGF- in immunocompromised patients and rabbits with CDC is an important local event, inhibiting effective cell-mediated immunity while permitting ineffective granuloma formation in response to C. albicans infection. One must therefore consider the complex role of this molecule and, specifically, a role for the beneficial effects of TGF- on Th-cell differentiation. Finally, our study provides evidence that TGF- production is at least a consequence of C. albicans infection and, indeed, could be a contributing factor to an unusual type of infection in the severely immunocompromised host. Other factors, such as host genetic profiles and prior immunosuppression, certainly have to be considered in a comprehensive understanding of the pathophysiology of CDC (19).