Journal of Medical and Veterinary Mycology (1994), 32, Supplement 1,203 210

Cytokines in the host response to mycotic agents

J. W. M U R P H Y l, B. A. W U - H S I E H 2, L. M. S I N G E R - V E R M E S 3, A. F E R R A N T E 4, S. M O S E R 5, M. R U S S O 3, C. A. C. VAZ 3,

E. B U R G E R 3, V. L. G. C A L I C H 3, I. C. K O W A N K O 4, D. A. R A T H J E N 4, A. J. M A R T I N 4, R. P. BUCY s AND Q. C H E N 5

1 University of Oklahoma Health Sciences Center, Oklahoma City, OK, and 2UCLA, Los Angeles, CA, USA, 3University of Sao Paulo, Brazil," 4Women's and Children's

Hospital, North Adelaide, South Australia," and 5University of Alabama, Birmingham, AL, USA

Protection against mycotic diseases is highly dependent on adequate natural and immune host defence mechanisms [21, 22]. Generally, a combination of several host defensive measures is required for complete protection against a mycotic agent [22]. However, there is one host defence mechanism that seems to stand above all others in effectiveness in the clearance of most fungal pathogens. That defensive mechanism is the cell-mediated immune (CMI) response [21]. Disruption of any single protective mech- anism, with the exception of the CMI response, may leave the host only slightly more vulnerable to progressive fungal disease. However, ablation of the CMI response results in progressive and often fatal disease in the case of systemic mycoses. CMI responses, as are humoral immune responses and activation of natural effector cells to clear micro-organisms from the host, driven and regulated by cytokines.

Cytokines are protein molecules produced and secreted by various host cells, and they allow host cells to communicate with one another. For instance, natural effector cells will produce cytokines which enhance the induction and direct the type of immune responses produced toward an infectious agent. Immune cells, such as sensitized T-cells, produce cytokines which influence the maturation and expansion of other T-cells or B-cells, and T-cells produce cytokines which may activate natural effector cells to more effectively kill fungal cells. In addition, some cytokines are involved in mediating the inflammatory and granulomatous responses which are generally a prerequisite to the clearance of micro-organisms or to restricting the micro-organism to a specific tissue(s). Studies on cytokines in systemic mycotic diseases are in the early stages and are still rather fragmentary. It is anticipated that once our knowledge of the role of the various cytokines in normal host protection against fungi is sufficient, then the development of immunomodulatory or immunoreplacement therapy in individuals suffering from a life- threatening mycotic disease can proceed. In this symposium, studies were presented that show representative findings currently available on cytokine production and function in paracoccidioidomycosis, histoplasmosis, candidiasis and blastomycosis.

Resistance mechanisms to Paracoccidioides brasiliensis experimental infection: a proposed model

By comparing the host's natural and acquired immune resistance responses to P. brasiliensis in two inbred strains of mice, one resistant to P. brasiliensis and the other

Correspondence address: Dr J. W. Murphy, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

203

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204 ~ U R P H ' Z Br AL.

Intense B-cell

activation

P. bras/7/ensls / .yeasts IL-IO y-IgN

----., /

Pb r gene Poor B-cell

activation

Progressive disease

T IL-4, 5, 6 Impaired

mocrophoge IL-I0 activation

y- IFN Efficient mocrophage

IL-2 activation

l Resolution

of the disease

FIG. 1. Hypothesis: immune regulatory mechanisms involved in experimental murine paracoccidioidomy- cosis. Resistant (A/Sn) mice carry the Pb r gene and handle the immune response to a Thl mode of activation leading to resolution of the disease. Susceptible (B10.A) mice carry the Pb S gene and mount a predominantly Th2 type of immune response resulting in progressive disease.

susceptible. L. M. Singer-Vermes, M. Russo, C. A. C. Vaz, E. Burger & V. L. G. Calich from Brazil have proposed a model to explain resistance and susceptibility based on the host's immune responses to P. brasiliensis. P. brasiliensis-susceptible B10.A mice infected intraperitoneally (IP) with 5 x 106 p. brasiliensis yeast cells display inefficient macrophage activation, transient expression of class II major histocompatibility complex (MHC) antigens by adherent peritoneal cells, depressed delayed-type hyper- sensitivity (DTH) responses, high levels of P. brasiliensis-specific antibodies (mainly of the IgG1, IgG2b, and IgA isotypes), very evident polyclonal activation of IgGl-, IgG2b-, and IgG2a-producing B-cells, and progressive paracoccidioidomycosis [5]. These findings suggest that in the susceptible mouse strain a predominant T-helper-2 (Th2) type of immune response develops in response to P. brasiliensis (Fig. 1). In contrast, A/Sn mice which are resistant to P. brasiliensis infection, respond to an IP injection with 5 x 106 viable P. brasiliensis yeast cells with the sustained expression of MHC class II antigens on adherent peritoneal cells, by developing a strong DTH response to P. brasiliensis and by displaying activated macrophages. The A/Sn mice produce only low levels of P. brasiliensis-specific antibodies which are IgG1 and IgG2a isotypes and show no evidence of polyclonal B-cell activation [5]. Thus, it appears that the A/Sn mice mount a Thl immune response during infection with P. brasiliensis (Fig. 1). From these findings, one might predict that the immune cells and cytokines produced in the A/Sn mice in response to a P. brasiliensis infection are the protective components of the immune response; whereas, the cells and cytokines produced in the B10.A mice exacerbate an infection with P. brasiliensis. It is well established in other infectious disease models that Thl cells are responsible for the CMI response, as shown by positive DTH reactions to the specific antigen [2, 6, 15, 27, 29]. The predominant

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C Y T O K I N E S IN T H E H O S T R E S P O N S E 205

cytokines produced by Thl cells are interferon-gamma (IFNy) and interleukin (IL)-2 [6, 13, 27]. Furthermore, it is known that IFN~, inhibits the proliferation of Th2 cells which are necessary for B-cell maturation to immunoglobulin secreting plasma cells [13]. Moreover, IFNy has been shown to activate macrophages to have increased fungicidal activity against P. brasiliensis [4]. In contrast to A/Sn mice, B10.A mice (in response to infection with P. brasiliensis) would be expected to produce cytokines such as IL-4 and IL-10 which are associated with antibody production [20]. IL-10 has been shown to block cytokine production by Thl cells [19]. Since Thl cells appear to be important in protecting against P. brasiliensis in the A/Sn mice, blocking Thl cell production by over-production of IL-4 and IL-10 in the B10.A mouse could account for the increased susceptibility of the B10.A mice to P. brasiliensis infection.

Tumour necrosis factor alpha (TNFa), primarily a product of macrophages, has the potential to activate other natural effector cells to have increased fungicidal activity [1, 7, 9, 31]. TNFa activity was not detected in the serum of B10.A or A/Sn mice at 1, 8 or 16 weeks after an IP or intravenous (IV) infection with P. brasiliensis. The lack of detectable amounts of TNF in the sera of infected mice does not exclude the possibility that there may be a sufficient local production of TNF to affect the disease process. The model proposed from these findings with P. brasiliensis resistant and susceptible mice (Fig. 1) might be a useful guide for further studies on cytokines with P. brasiliensis and for investigations with other dimorphic fungal pathogens such as Blastomyces dermatitidis, Coccidioides immitis and Histoplasma capsulatum.

Modulation of IFN 7 and TNF~t in histoplasmosis

Cytokine production and its relationship to disease regression or progression has also been studied in a murine model of histoplasmosis by B. Wu-Hsieh at the UCLA School of Medicine in Los Angeles, California. In contrast to P. brasiliensis, H. capsulatum, the aetiological agent of histoplasmosis, is an intracellular parasite of mononuclear phagocytes. The fungus disseminates to different organ systems after IV infection. To understand the kinetics of cytokine production and disease regression, C57BL/6 mice were given a sublethal dose of H. capsulatum yeast cells IV. Disease progression in the mice was monitored by a tissue census of H. capsulatum and cytokine production by spleen cells from the mice. Disease progression followed a similar pattern in animals infected with either 2 × 104 or 2 x 105 yeast cells, both of which are sublethal doses. The cytokines TNFa and IFNy were monitored throughout the course of the study and the cytokines were produced at levels that coincided with the fungal burden. Cytokine production in the infected mice appeared to be tightly regulated according to the progression of disease; however, TNFa and IFNy were independently regulated. This was evident from the fact that the depletion of one cytokine in infected mice by the use of an antibody to the cytokine did not affect the production of the other cytokine. From the cytokine depletion results, it was apparent that endogenously produced TNFa and IFN7 are both important in the clearance of H. capsulatum yeast cells in the infected host. Depletion of either or both TNFa or IFNy exacerbated the histoplasmosis, and animals died. It has previously been shown that IFN~, alone activated murine peritoneal macrophages but did not fully activate spleen cells to inhibit the replication of H. capsulatum [31]. However, IFNy in conjunction with lipopolysaccharide activates splenic macrophages in vitro to more effectively inhibit the replication of H. capsulatum [31]. The importance of IFN7 in host clearance of H. capsulatum yeast cells was studied in two mouse strains, A/J and C57BL/6. AJJ mice were found to be more efficient at

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206 MURPHY E T A L .

clearing H. capsulatum than were C57BL/6 mice [30]. IFN7 production by spleen cells from these two mouse strains followed a similar pattern except at day 3 of infection. At that time, the earliest time when IFN7 could be detected, A/J mice produced significantly greater amounts of IFNy than C57BL/6 mice [30]. Recent data from immunocytochemical studies show that IL-4 was produced in the spleens of C57BL/6 mice as early as 7 days after infection. IL-4 was also produced in the spleens of A/J mice, but later in the course of infection. It appears that the temporal sequence of IFN7 and IL-4 production governs the efficiency of host macrophages to clear H. capsulatum from tissues.

From antibody depletion studies, it appears that endogenously produced TNFa is essential for the effective clearance of H. capsulatum. However, the mechanism by which TNFa aids clearance in histoplasmosis has not been clearly defined. TNFa did not activate peritoneal macrophages, nor did it activate splenic macrophages, when used in combination with IFNT. These results suggest that TNFa may function to protect against H. capsulatum in ways other than by activation of macrophages. In experimen- tal bacterial infections, TNFa has been shown to be involved in granuloma formation in the liver of infected animals [14]. TNFa may play a similar role in experimental histoplasmosis. Furthermore, evidence from immunocytochemical studies shows that TNF~ may also regulate IL-6 production. When the production of TNFa gradually diminished during the course of infection, IL-6 was actively produced. IL-6 is known to be a cytokine that plays an important role in the terminal differentiation of B-cells into plasma cells. Thus, TNFa indirectly regulates the production of antibodies via IL-6. This notion is supported by data from studies on Histoplasma-specific antibodies.

Antifungal activity of TNF-activated neutrophils TNFa has multiple biological properties which include the activation of neutrophils. TNF-activated neutrophils show increased phagocytosis, increased release of oxygen- derived reactive species and increased release of constituents from both specific and azurophilic granules [10, 16, 23]. The antifungal capabilities of TNF-activated neutro- phils have been investigated by A. Ferrande, I. C. Kowanko, D. A. Rathjen & A. J. Martin at the University of Adelaide. Tissues infected with fungi show an accumulation of neutrophils and neutrophil infiltrates are a hallmark of Candida lesions. Furthermore, there is a correlation between increased susceptibility to Candida infection and neutropenia [8]. Neutrophils have been shown to kill C albicans in vitro and treatment with TNF results in increased neutrophil killing of C albicans and Candida glabrata blastoconidia [7, 9]. Together these facts indicate that neutrophils are important cells in host defence against C albicans.

Neutrophils have two TNF receptors on their surface, a 75 kDa and a 55 kDa peptide [3, 12]. These receptors bind both TNFa and TNFfl, and ligation of these receptors with TNF leads to priming of the neutrophils for increased surface expression of complement receptor type 3 (CR3) and enhanced responses to complement opsonized fungi [16, 18]. The mechanism by which TNF increases the antifungal activity of neutrophils may be partly related to an increase in the expression of CR3 [16, 18] which promotes the interaction of the fungi with neutrophils, thereby enhancing the release of both oxygen-derived reactive species and granule enzymes [9]. The enhance- ment of H202 release and myeloperoxidase from azurophilic granules, promotes the killing of microbes through the generation of hypochlorite [17].

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CYTOKINES IN THE HOST RESPONSE

TABLE 1. Properties of the small synthetic TNF peptide, 419

Property TNF

Activity

419 (Region 70-80)

Tumour cell cytotoxicity + + + Tumour regression + + + Endothelial PCA activity + + +

induces expression of E-selectin, ICAM, VCAM

Neutrophil activation + + + + + +

PCA; pro-coagulant activity. ICAM; intercellular adhesion molecules. VCAM; vascular cellular adhesion molecules.

207

TNF receptors on neutrophils are readily shed following stimulation with a number of soluble stimuli [25, 28]. When neutrophils are pre-treated with either opsonized bacteria or opsonized C glabrata, the neutrophils show decreased TNF binding [12]. A similar phenomenon was observed when neutrophils were incubated with opsonized C. albicans blastoconidia but did not occur when opsonized sheep erythrocytes replaced the opsonized C. albicans. These findings imply that microbial products, by interacting with neutrophils, can depress the ability of TNF to enhance neutrophil antifungal properties. It is also well established that the extracellular domains of both TNF receptors are released and can be detected in biological fluids during a variety of diseases [24]. The released TNF receptors can bind TNF and prevent TNF interactions with the TNF receptors on cells. It has been shown previously that the neutrophil stimulating activity of TNF can be prevented by the 33 kDa TNF receptor fragment isolated from biological fluids [11].

If the TNF enhancement of fungal killing could be exploited for therapeutic purposes this may be useful in the treatment of infections with C. albicans or C. glabrata. Some avenues have been identified to pursue this idea. TNF is highly toxic, so its use in enhancing antimicrobial immunity has met with limitations. One approach that has been taken to circumvent this limitation has been to identify regions of the TNF molecule which have neutrophil stimulating activity but lack the typical toxicity inducing regions [26]. Small peptides spanning residues 70-80 were synthesized. These small peptides retained the neutrophil activation and priming properties but lacked the ability to induce endothelial cell pro-coagulant activity and cell adhesion molecule expression (E-selectin, ICAM, VCAM). They were relatively non-toxic when injected into experimental animals. A summary of activities of one of the peptides is shown in Table 1. The peptide was capable of enhancing the neutrophil respiratory burst induced by fMLP, suggesting that the peptide may be therapeutically useful for enhancing the antimicrobial properties of neutrophils.

Role of cytokines in the pathogenesis of experimental murine blastomycosis

The role of cytokines in the pathogenesis of experimental murine blastomycosis has been the focus of studies by S. A. Moser, R. P. Bucy& Q. Chen at the University of

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208 MURPHY E T A L .

Alabama in Birmingham. BALB/cByJ mice were given (via the intratracheal route) 104 viable conidia of either B. dermatitidis strain CR (virulent) or FW (avirulent), or H. capsulatum G217B. At varying times following inoculation, lungs were removed and total RNA was extracted. The mRNAs for the cytokines, IL-lfl, IL-2, IL-4, IL-5, IL-6, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), TNFa, TNFfl, IFNT, inducible nitric oxide synthase (iNOS), and macrophage inflammatory protein-1 a (MIP-la) were measured using reverse transcription and polymerase chain reaction (RT-PCR) amplification. In addition to total lung analysis, alveolar lavage macro- phages were isolated and analysed for cytokine mRNAs following exposure to the conidia of B. dermatitidis or H. capsulatum.

It is apparent from the results of these studies that mice given virulent B. dermatitidis differ from control animals in that there was a strong, yet transient IFN7 response in the infected mice. In contrast, the mice given the avirulent B. dermatitidis isolate did not display increased IFN~ mRNA and differed from the controls in that there was a late induction of MIP-la and TNFa mRNA. Virulent 1t. capsuIatum induced a pattern of cytokine mRNA similar to that seen in mice given virulent B. dermatitidis. Evaluations with in situ hybridization were performed for the cytokines of interest at time points which were identified by the RT-PCR measurements as being important. The cytokines induced were localized to the pulmonary lesions and the strongest staining reactions were seen with TNFa and iNOS in B. dermatitidis-infected mouse lungs. In comparison, increased cytokine mRNA in response to H. capsulatum included IFNT, TNFa, iNOS, IL-lfl, MIP-la and IL-6. These data suggest that macrophages are the primary cell activated in both infections with B. dermatitidis and H. capsulatum, and that the RT-PCR approach will be helpful in defining the divergence between the histological responses to B. dermatitidis and H. capsulatum.

Summary In summary, different approaches have been taken to understand cytokine responses to different fungal infections. Singer-Vermes and co-investigators indirectly examined cytokine responses to paracoccidioidomycosis by studying the types of cellular and humoral immune responses that were induced in resistant and susceptible mouse strains. Their results implicated Thl cell responses in the resistant mouse strain and Th2 cell responses in the mouse strain susceptible to paracoccidioidomycosis. By measuring cytokine production and through cytokine depletion experiments, Wu-Hsieh showed that besides IFNT, TNFa was important in host defences against the intracellular pathogen, H. capsulatum. Both cytokines play important roles in the regulation of other cytokines. In histoplasmosis, the dynamics of the complex interactions amongst cytokines govern the efficiency of host clearance of the fungus from tissues. Ferrante and collaborators, examining TNFa and TNFa receptors on neutrophils presented data showing that TNFa plays an important role in the activation of neutrophils for anti-Candida activity. Through the detection of cytokine mRNAs with RT-PCR, Moser and co-workers found that cytokine mRNAs of macrophage origin were produced preferentially in the lungs of mice infected with Histoplasma or Blastomyces.

A great challenge still lies ahead of us. It is well understood that the interactions of cytokines are extremely complex at the levels of the induction and expression of the immune responses as well as on effects on natural cellular defences. Work accomplished thus far has laid the ground work for future studies in the effort to dissect host cytokine

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C Y T O K I N E S IN THE H O S T R E S P O N S E 209

responses and to understand the roles of cytokines in protection against fungal infections.

C O N T R I B U T O R S

The contributors to this symposium were: L. Singer-Vermes, M. Russo, C. A. C. Vaz, E. Burger & V. L. G. Calieh, Resistance mechanisms to Paracoccidioides brasiliensis experimental infection." a proposed model; B. Wu-Hsieh, Modulation of lFN-gamma and TNF-alpha in histoplasmosis; A. Ferrante, I. C. Kowanko, D. A. Rathjen & A. d. Martin, Antifungal activity of TNF-activated neutrophils; S. A. Moser, R. P. Buey & Q.-Y. Chen, Role of cytokines in the pathogenesis of experimental murine blastomycosis. The co-convenors were J. W. Murphy and B. Wu-Hsieh.

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