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Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice
FEMS Immunology and Medical Microbiology 26 (1999) 299^307
Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice Bart Jan Kullberg, Mihai G. Netea, Alieke G. Vonk, Jos W.M. van der Meer Department of Medicine, Catholic University Nijmegen and University Hospital, Nijmegen, The Netherlands Received 16 March 1999 ; revised 23 July 1999; accepted 28 July 1999
Abstract Neutrophils (PMNs) constitute the main mechanism of host defense against acute invasive and disseminated candidiasis. Recent studies have demonstrated that tumor necrosis factor-K (TNFK), interleukin-6 (IL-6) and granulocyte colonystimulating factor (G-CSF) play an important role in the recruitment of PMNs at the site of invasive Candida infection. In the absence of either TNFK or IL-6, the course of experimental disseminated candidiasis is more severe, due to defective PMN recruitment. Treatment of mice with recombinant G-CSF (rG-CSF) leads to a significantly reduced mortality during disseminated candidiasis. The outgrowth of Candida albicans from the organs of rG-CSF-treated mice is significantly decreased. Treatment with the combination of rG-CSF and fluconazole has an additive effect on the reduction of fungal load in the organs. In subacute or chronic disseminated Candida infection, rG-CSF is less effective, indicating that neutrophil recruitment and activation are crucial in acute, life-threatening candidiasis, whereas other host defense mechanisms control the outcome of less overwhelming invasive Candida infection. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Granulocyte colony-stimulating factor ; Tumor necrosis factor; Fluconazole ; Neutrophil; Candida albicans
1. Introduction Disseminated candidiasis is increasingly common in patients with a variety of underlying diseases [1]. Recently, much progress has been made in the development of anti-fungal therapy. New classes of antifungal drugs have shown increased e¤cacy and fewer side e¡ects [2]. Despite these developments, treatment failure still is a signi¢cant problem, occurring in 20^30% of patients with disseminated candidiasis [3,4]. In speci¢c groups of patients, such as those with persistent neutropenia, failure rates are even higher [5]. Resolution of these infections is often dependent on reconstitution of granulocyte function
and there is abundant evidence that polymorphonuclear leukocytes (PMNs) constitute the main mechanism of host defense against invasive and disseminated candidiasis [6]. Therefore, immunotherapy aimed at enhancing both the number and function of neutrophils may prove extremely productive in this ¢eld, even in non-neutropenic hosts. Several strategies may be applied to enhance the anti-fungal activity of PMNs against an invasive Candida infection. First, increasing the production of PMNs in the bone marrow and the circulating numbers of peripheral blood PMNs may be bene¢cial. Obviously, this approach is particularly helpful in case of chemotherapy-induced neutropenia [5].
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Recently however, we have demonstrated that increasing the numbers of circulating PMNs exceeding the apparently `normal' range signi¢cantly improves the outcome of disseminated candidiasis in non-neutropenic patients [7]. The second target of immune intervention may be the local recruitment of PMNs at the site of infection. This stage includes adherence of PMNs to the vascular endothelial cells, their migration through the vessel wall and subsequent granuloma formation, leading to containment of the infection. Third, phagocytosis of Candida blastospores by PMNs may be targeted by immunotherapy. This process is under the control of several cytokines, as has been demonstrated by in vitro studies [8,9]. Fourth, PMNs constitute the main e¡ector cells in both intracellular killing of blastospores and extracellular damage to Candida hyphae and pseudohyphae and several cytokines playing a role in this process have been identi¢ed [10^12].
2. Cytokines and the recruitment of neutrophils at the site of infection The in£ux of PMNs at the site of an invasive Candida infection is controlled by a variety of endogenous pro-in£ammatory cytokines. The role of interleukin-6 (IL-6) in the activation of neutrophils is poorly understood, although studies performed in mice in which the gene for IL-6 has been disrupted (IL-63/3 mice) have suggested a defective neutrophil function in the absence of the cytokine [13]. Recent studies have elucidated the role of IL-6 in host defense against experimental Candida albicans infection. Cell-wall components of C. albicans stimulate IL-6 production in vitro [14] and elevated IL-6 concentrations have been detected during experimental infection with C. albicans [15]. Moreover, it has been demonstrated that IL-63/3 mice are more susceptible to infection with C. albicans [16]. The importance of PMNs in the IL-6-mediated resistance to candidiasis has become clear from experiments in neutropenic IL-63/3 mice. These mice do not di¡er in their susceptibility to disseminated candidiasis compared with neutropenic control mice that do produce IL-6, indicating that PMNs are likely to be the dominant protective mechanism through which IL-6 exerts its bene¢cial e¡ects [17]. Indeed,
we found that the recruitment of neutrophils into the peritoneal cavity of IL-63/3 mice with a C. albicans peritonitis was signi¢cantly reduced. In contrast, the killing mechanisms of PMNs of IL-63/3 mice against Candida are intact [17]. Conversely, the administration of recombinant IL-6 decreases the fungal load in the organs of mice with disseminated candidiasis [16]. It is believed that the dominance of either of the two T-helper subsets (Th1 and Th2) closely correlates with the outcome of Candida infection [18]. In models of disseminated candidiasis, occurrence of Th1 responses is associated with protection, whereas Th2 responses correlate with progressive infection [19]. IL-6 is among the cytokines that are required to induce a Th1 response and absence of IL-6 during disseminated candidiasis leads to a reduced production of IL-12 and an increased IL-10 release [16]. This Th2-type pattern has been shown to adversely a¡ect the outcome of experimental disseminated candidiasis, as administration of exogenous rIL-10 leads to increased mortality to Candida infection, whereas administration of rIL-12 protects mice against disseminated candidiasis [20,21]. Thus, the regulation of neutrophil recruitment and subsequent development of cellular immunity appear to be closely related. The pro-in£ammatory cytokine tumor necrosis factor-K (TNFK) is also induced by Candida mannoproteins in vitro [14]. Its requirement for the host defense has become clear from experiments in which treatment of mice with monoclonal antibodies against TNFK enhances the mortality to experimental disseminated candidiasis [15,22]. Likewise, treatment with pharmacological inhibitors of TNFK production leads to enhanced mortality and increased outgrowth of C. albicans during disseminated candidiasis in mice [23]. Non-neutropenic mice which lack the genes encoding TNFK and L (TNF3/3) are highly susceptible to disseminated candidiasis [24]. In contrast, neutropenic TNF3/3 mice do not di¡er from neutropenic control animals in terms of host resistance to candidiasis [25]. The recruitment of PMNs at the site of a localized Candida infection is impaired in TNF3/3 mice [25], whereas similar killing of Candida blastospores was reported for neutrophils and macrophages of TNF3/3 mice [24], suggesting that recruitment of PMNs at the site of infection has been a major factor for the increased
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Fig. 1. Survival of mice pretreated with rG-CSF 24 h before injection of 5U105 CFU of C. albicans. Mice (10 animals per group) were injected with a single dose of either 200 or 500 ng of rG-CSF or saline, 1 day before infection. Signi¢cant di¡erence between controls and treatment groups: P 6 0.005 (Kaplan-Meier log-rank test).
susceptibility to disseminated candidiasis of non-neutropenic knock-out mice de¢cient in TNF. Fas ligand (FasL), a member of the TNF family, also plays a role in regulating neutrophil recruitment and host defense to candidiasis [26,27]. An other cytokine involved in PMN in£ux is IL-1 [28], although its exact role in invasive candidiasis has not been established [29]. In addition, the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) are undoubtedly among the most important mediators of PMN recruitment during infection [30].
3. G-CSF and experimental candidiasis G-CSF is a hematopoietic growth factor that promotes the proliferation and di¡erentiation of neutrophils from bone marrow. The recombinant form (rG-CSF) has widespread clinical use in chemotherapy-induced neutropenia, severe chronic neutropenia, peripheral blood cell mobilization and bone marrow transplantation [31]. rG-CSF not only increases the numbers of PMNs, but also increases their recruitment at the site of infection [30] and enhances their capacity for killing Candida blastospores and pseudohyphae in vitro [9].
Previous studies have demonstrated a bene¢cial e¡ect on the course of experimental infection with C. albicans when rG-CSF was administered prophylactically to prevent neutropenia [32,33]. Recently, we have shown that recombinant murine G-CSF also bene¢cially in£uences the course of potentially lethal acute disseminated candidiasis in non-neutropenic CBA mice [34]. Pretreatment with a single subcutaneous dose of 500 ng of murine rG-CSF reduced the mortality (Fig. 1) and signi¢cantly decreased the outgrowth of C. albicans in the organs of the animals (Fig. 2A). Also, we were able to demonstrate that PMNs were mobilized by rG-CSF at the site of the infection and that their production of oxygen radicals was increased [34]. Histopathology showed extensive hyphal outgrowth of C. albicans as well as yeast forms in the non-treated animals and remarkably few PMNs could be found at the sites of infection. In animals that had received a single dose of rG-CSF, the infectious foci were less numerous than in controls and the in£ammatory in¢ltrates contained larger amounts of PMNs, with fewer yeasts present. In contrast to those in placebo-treated controls, Candida cells in the organs of rG-CSF-treated animals were almost completely limited to the yeast form and in most in¢ltrates, hyphae were absent [34]. In subsequent studies, we have investigated whether rG-CSF is e¡ective when given at later
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Fig. 2. Outgrowth of C. albicans in the kidneys and liver of mice after intravenous (i.v.) injection of 5U105 CFU of C. albicans. Mice were injected subcutaneous (s.c.) with 500 ng of rG-CSF (F) or with saline (E) on day 31 (A) or on day +1 of infection (B) or with daily injections from day +1 through day +10 of infection (C). Each column represents the mean þ S.E.M. for 9^15 animals. Signi¢cant di¡erences between rG-CSF-treated mice and control mice are indicated (*, P 6 0.05; ** P 6 0.001; Mann-Whitney U test).
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Fig. 3. Histopathology of the kidneys of mice 3 days after i.v. injection of 5U105 CFU of C. albicans. In control animals, in¢ltrates are numerous and consist of large amounts of C. albicans both in the yeast form and as pseudohyphae. Only a moderate in£ux of PMNL is found (A). Administration of rG-CSF as late as 24 h after infection resulted in a reduced outgrowth of pseudohyphae as well as an enhanced in£ux of PMNL at the sites of infection, as compared to control animals (B). Periodic acid Schi¡ (PAS) staining; original magni¢cation, 25U.
time points in models of subacute and less overwhelming disseminated candidiasis or when combined with anti-fungal drugs. In the model of rapidly lethal Candida infection, the e¡ect of a single dose of rG-CSF was greatest when given 24 h before injection of C. albicans. When treatment was postponed until 6 h after the onset of infection, the bene¢cial
e¡ect on the survival and organ load of Candida became less pronounced and if administration of rG-CSF was delayed until 24 h after injection of large amounts of C. albicans, a signi¢cant e¡ect on neither mortality nor the outgrowth of microorganisms was found (Fig. 2B) [34]. Nevertheless, such a treatment, even when begun as late as after 24 h, had
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Fig. 4. Survival of mice treated with 500 ng of rG-CSF 6 h after injection of 5U105 CFU of C. albicans, £uconazole (10 mg kg31 bid) or both. Mice (30 animals per group) were injected with a single dose of rG-CSF or saline 1 day before infection. Signi¢cant di¡erence between controls and G-CSF-treated (P 6 0.05) or £uconazole treatment groups (P 6 0.001; Kaplan-Meier log-rank test).
a notable impact on the histopathology at the sites of infection. A signi¢cantly reduced outgrowth of pseudohyphae as well as an enhanced in£ux of PMNs at the sites of infection was seen on microscopic examination in rG-CSF-treated mice, when compared with placebo-treated controls (Fig. 3). When more attenuated or sublethal types of disseminated Candida infection were studied, treatment with rG-CSF for up to 10 days had little e¡ect. The outgrowth of C. albicans in rG-CSF-treated animals did not di¡er signi¢cantly from that in untreated controls (Fig. 2C). This is in agreement with the observations of others, who described a bene¢cial e¡ect on the survival of mice only when rG-CSF was either given in very high doses [35] or in combination therapy with anti-fungal drugs, but only when begun before or immediately after infection and only when small amounts of C. albicans were injected [36,37].
4. Combined therapy with rG-CSF and £uconazole Although the results of treatment of experimental candidiasis with rG-CSF are encouraging, it may be more clinically relevant to determine whether rGCSF can augment anti-fungal drug treatment rather
than stand on its own. As an anti-fungal drug, we selected £uconazole, because it has been shown to be the drug of choice for treatment of candidemia and disseminated infection with C. albicans [3,38]. At 6 h after the onset of experimental disseminated candidiasis in non-neutropenic mice, treatment was started with either rG-CSF, £uconazole or both. In a potentially lethal model of disseminated candidiasis, the mortality was signi¢cantly reduced by treatment with rG-CSF alone, as well as by £uconazole in a dose-dependent fashion (Fig. 4). Although combined therapy appeared not to have an additive e¡ect on survival, the e¡ect of both drugs was additive in inhibiting the fungal burden in the organs of infected mice. Treatment with £uconazole alone led to a signi¢cantly reduced outgrowth of Candida in the kidneys of mice (P 6 0.005), whereas a similar trend was seen in the liver (Fig. 5). The combination of rGCSF and £uconazole signi¢cantly further reduced the numbers of colony forming units (CFU) in both the liver and the kidneys (P 6 0.01) when compared with £uconazole treatment only (Fig. 5). Such an additive e¡ect of rG-CSF and conventional antifungal therapy is in agreement with earlier ¢ndings [35]. Remarkably, whereas the additive e¡ect of rGCSF and £uconazole was limited to reducing the outgrowth of Candida, combined therapy with rG-
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Fig. 5. Outgrowth of C. albicans in the kidneys and liver of mice after i.v. injection of 5U105 CFU of C. albicans. Mice were injected s.c. with 500 ng of rG-CSF or with saline 6 h after the onset of infection, treated with £uconazole (F) at 2.5 or 10 mg kg31 bid or with rGCSF and £uconazole. Each column represents the mean þ S.E.M. for six animals. Signi¢cant di¡erences (P 6 .05) are indicated between control mice and treatment groups (*) and between mice treated with corresponding doses of £uconazole with and without rG-CSF (2, Mann-Whitney U test).
CSF and amphotericin B bene¢cially in£uenced survival of mice [35]. In a model of chronic, non-lethal disseminated candidiasis, prolonged therapy with rG-CSF (500 ng bid) for up to 10 days was investigated, in combination with £uconazole for 3 days. In this model, £uconazole signi¢cantly reduced the outgrowth of Candida CFU during the course of treatment, but rG-CSF did not produce an additional e¡ect (data not shown). From these experiments, it is suggested that rGCSF immunotherapy of experimental disseminated candidiasis in mice is most e¡ective during severe progressive disseminated infections. As was suggested by the paucity of invading PMNs in the infected organs of untreated non-neutropenic mice [34], the local PMN response may be the factor limiting host resistance under those circumstances and exogenous rG-CSF is crucial to in£uence the course of infection.
Although treatment with rG-CSF during less overwhelming models of candidiasis leads to enhanced PMN recruitment and prevention of hyphal outgrowth, the clinical course of infection was not a¡ected. This may indicate that, in subacute infections, PMNs no longer play the pivotal role in host defense, which ultimately determines the outcome of infection. Other mechanisms, such as cell-mediated immunity, are of importance at the later stages of infection. In these types of infection, induction of a Th1-type cellular response, associated with the production of IL-1, TNFK, interferon (IFN)-Q and IL-12, has been show to confer protection against C. albicans infection [21]. The role of PMNs in chronic candidiasis may be primarily as a source of pro-in£ammatory cytokines, since these cells have been shown to produce TNFK, IL-6 and IL-12 upon stimulation with virulent Candida strains, but not non-virulent, agerminative Candida blastospores [39].
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5. Conclusion In conclusion, it has become clear that production of PMNs, their migration to the site of infection, their capacity for killing Candida hyphae and blastospores, as well as their role as producers of pro-in£ammatory cytokines is substantial in the host defense against acute and life-threatening disseminated candidiasis. Experiments in knock-out mice as well as with recombinant cytokines have demonstrated that TNFK, IL-6 and G-CSF are among the mediators that direct the activation and recruitment of PMNs to control invasive Candida infections.
Acknowledgements This work was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy (San Diego, CA, USA), 1998. Part of these studies were supported by an unrestricted grant from Amgen. B.J. Kullberg was supported by a grant from the Royal Netherlands Academy of Arts and Sciences. The experiments were approved by the ethics committee for animal experiments at the Catholic University Nijmegen.
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Modulation of neutrophil function in host defense against disseminated Candida albicans infection in mice Bart Jan Kullberg, Mihai G. Netea, Alieke G. Vonk, Jos W.M. van der Meer Department of Medicine, Catholic University Nijmegen and University Hospital, Nijmegen, The Netherlands Received 16 March 1999 ; revised 23 July 1999; accepted 28 July 1999
Abstract Neutrophils (PMNs) constitute the main mechanism of host defense against acute invasive and disseminated candidiasis. Recent studies have demonstrated that tumor necrosis factor-K (TNFK), interleukin-6 (IL-6) and granulocyte colonystimulating factor (G-CSF) play an important role in the recruitment of PMNs at the site of invasive Candida infection. In the absence of either TNFK or IL-6, the course of experimental disseminated candidiasis is more severe, due to defective PMN recruitment. Treatment of mice with recombinant G-CSF (rG-CSF) leads to a significantly reduced mortality during disseminated candidiasis. The outgrowth of Candida albicans from the organs of rG-CSF-treated mice is significantly decreased. Treatment with the combination of rG-CSF and fluconazole has an additive effect on the reduction of fungal load in the organs. In subacute or chronic disseminated Candida infection, rG-CSF is less effective, indicating that neutrophil recruitment and activation are crucial in acute, life-threatening candidiasis, whereas other host defense mechanisms control the outcome of less overwhelming invasive Candida infection. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Granulocyte colony-stimulating factor ; Tumor necrosis factor; Fluconazole ; Neutrophil; Candida albicans
1. Introduction Disseminated candidiasis is increasingly common in patients with a variety of underlying diseases [1]. Recently, much progress has been made in the development of anti-fungal therapy. New classes of antifungal drugs have shown increased e¤cacy and fewer side e¡ects [2]. Despite these developments, treatment failure still is a signi¢cant problem, occurring in 20^30% of patients with disseminated candidiasis [3,4]. In speci¢c groups of patients, such as those with persistent neutropenia, failure rates are even higher [5]. Resolution of these infections is often dependent on reconstitution of granulocyte function
and there is abundant evidence that polymorphonuclear leukocytes (PMNs) constitute the main mechanism of host defense against invasive and disseminated candidiasis [6]. Therefore, immunotherapy aimed at enhancing both the number and function of neutrophils may prove extremely productive in this ¢eld, even in non-neutropenic hosts. Several strategies may be applied to enhance the anti-fungal activity of PMNs against an invasive Candida infection. First, increasing the production of PMNs in the bone marrow and the circulating numbers of peripheral blood PMNs may be bene¢cial. Obviously, this approach is particularly helpful in case of chemotherapy-induced neutropenia [5].
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Recently however, we have demonstrated that increasing the numbers of circulating PMNs exceeding the apparently `normal' range signi¢cantly improves the outcome of disseminated candidiasis in non-neutropenic patients [7]. The second target of immune intervention may be the local recruitment of PMNs at the site of infection. This stage includes adherence of PMNs to the vascular endothelial cells, their migration through the vessel wall and subsequent granuloma formation, leading to containment of the infection. Third, phagocytosis of Candida blastospores by PMNs may be targeted by immunotherapy. This process is under the control of several cytokines, as has been demonstrated by in vitro studies [8,9]. Fourth, PMNs constitute the main e¡ector cells in both intracellular killing of blastospores and extracellular damage to Candida hyphae and pseudohyphae and several cytokines playing a role in this process have been identi¢ed [10^12].
2. Cytokines and the recruitment of neutrophils at the site of infection The in£ux of PMNs at the site of an invasive Candida infection is controlled by a variety of endogenous pro-in£ammatory cytokines. The role of interleukin-6 (IL-6) in the activation of neutrophils is poorly understood, although studies performed in mice in which the gene for IL-6 has been disrupted (IL-63/3 mice) have suggested a defective neutrophil function in the absence of the cytokine [13]. Recent studies have elucidated the role of IL-6 in host defense against experimental Candida albicans infection. Cell-wall components of C. albicans stimulate IL-6 production in vitro [14] and elevated IL-6 concentrations have been detected during experimental infection with C. albicans [15]. Moreover, it has been demonstrated that IL-63/3 mice are more susceptible to infection with C. albicans [16]. The importance of PMNs in the IL-6-mediated resistance to candidiasis has become clear from experiments in neutropenic IL-63/3 mice. These mice do not di¡er in their susceptibility to disseminated candidiasis compared with neutropenic control mice that do produce IL-6, indicating that PMNs are likely to be the dominant protective mechanism through which IL-6 exerts its bene¢cial e¡ects [17]. Indeed,
we found that the recruitment of neutrophils into the peritoneal cavity of IL-63/3 mice with a C. albicans peritonitis was signi¢cantly reduced. In contrast, the killing mechanisms of PMNs of IL-63/3 mice against Candida are intact [17]. Conversely, the administration of recombinant IL-6 decreases the fungal load in the organs of mice with disseminated candidiasis [16]. It is believed that the dominance of either of the two T-helper subsets (Th1 and Th2) closely correlates with the outcome of Candida infection [18]. In models of disseminated candidiasis, occurrence of Th1 responses is associated with protection, whereas Th2 responses correlate with progressive infection [19]. IL-6 is among the cytokines that are required to induce a Th1 response and absence of IL-6 during disseminated candidiasis leads to a reduced production of IL-12 and an increased IL-10 release [16]. This Th2-type pattern has been shown to adversely a¡ect the outcome of experimental disseminated candidiasis, as administration of exogenous rIL-10 leads to increased mortality to Candida infection, whereas administration of rIL-12 protects mice against disseminated candidiasis [20,21]. Thus, the regulation of neutrophil recruitment and subsequent development of cellular immunity appear to be closely related. The pro-in£ammatory cytokine tumor necrosis factor-K (TNFK) is also induced by Candida mannoproteins in vitro [14]. Its requirement for the host defense has become clear from experiments in which treatment of mice with monoclonal antibodies against TNFK enhances the mortality to experimental disseminated candidiasis [15,22]. Likewise, treatment with pharmacological inhibitors of TNFK production leads to enhanced mortality and increased outgrowth of C. albicans during disseminated candidiasis in mice [23]. Non-neutropenic mice which lack the genes encoding TNFK and L (TNF3/3) are highly susceptible to disseminated candidiasis [24]. In contrast, neutropenic TNF3/3 mice do not di¡er from neutropenic control animals in terms of host resistance to candidiasis [25]. The recruitment of PMNs at the site of a localized Candida infection is impaired in TNF3/3 mice [25], whereas similar killing of Candida blastospores was reported for neutrophils and macrophages of TNF3/3 mice [24], suggesting that recruitment of PMNs at the site of infection has been a major factor for the increased
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Fig. 1. Survival of mice pretreated with rG-CSF 24 h before injection of 5U105 CFU of C. albicans. Mice (10 animals per group) were injected with a single dose of either 200 or 500 ng of rG-CSF or saline, 1 day before infection. Signi¢cant di¡erence between controls and treatment groups: P 6 0.005 (Kaplan-Meier log-rank test).
susceptibility to disseminated candidiasis of non-neutropenic knock-out mice de¢cient in TNF. Fas ligand (FasL), a member of the TNF family, also plays a role in regulating neutrophil recruitment and host defense to candidiasis [26,27]. An other cytokine involved in PMN in£ux is IL-1 [28], although its exact role in invasive candidiasis has not been established [29]. In addition, the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) are undoubtedly among the most important mediators of PMN recruitment during infection [30].
3. G-CSF and experimental candidiasis G-CSF is a hematopoietic growth factor that promotes the proliferation and di¡erentiation of neutrophils from bone marrow. The recombinant form (rG-CSF) has widespread clinical use in chemotherapy-induced neutropenia, severe chronic neutropenia, peripheral blood cell mobilization and bone marrow transplantation [31]. rG-CSF not only increases the numbers of PMNs, but also increases their recruitment at the site of infection [30] and enhances their capacity for killing Candida blastospores and pseudohyphae in vitro [9].
Previous studies have demonstrated a bene¢cial e¡ect on the course of experimental infection with C. albicans when rG-CSF was administered prophylactically to prevent neutropenia [32,33]. Recently, we have shown that recombinant murine G-CSF also bene¢cially in£uences the course of potentially lethal acute disseminated candidiasis in non-neutropenic CBA mice [34]. Pretreatment with a single subcutaneous dose of 500 ng of murine rG-CSF reduced the mortality (Fig. 1) and signi¢cantly decreased the outgrowth of C. albicans in the organs of the animals (Fig. 2A). Also, we were able to demonstrate that PMNs were mobilized by rG-CSF at the site of the infection and that their production of oxygen radicals was increased [34]. Histopathology showed extensive hyphal outgrowth of C. albicans as well as yeast forms in the non-treated animals and remarkably few PMNs could be found at the sites of infection. In animals that had received a single dose of rG-CSF, the infectious foci were less numerous than in controls and the in£ammatory in¢ltrates contained larger amounts of PMNs, with fewer yeasts present. In contrast to those in placebo-treated controls, Candida cells in the organs of rG-CSF-treated animals were almost completely limited to the yeast form and in most in¢ltrates, hyphae were absent [34]. In subsequent studies, we have investigated whether rG-CSF is e¡ective when given at later
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Fig. 2. Outgrowth of C. albicans in the kidneys and liver of mice after intravenous (i.v.) injection of 5U105 CFU of C. albicans. Mice were injected subcutaneous (s.c.) with 500 ng of rG-CSF (F) or with saline (E) on day 31 (A) or on day +1 of infection (B) or with daily injections from day +1 through day +10 of infection (C). Each column represents the mean þ S.E.M. for 9^15 animals. Signi¢cant di¡erences between rG-CSF-treated mice and control mice are indicated (*, P 6 0.05; ** P 6 0.001; Mann-Whitney U test).
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Fig. 3. Histopathology of the kidneys of mice 3 days after i.v. injection of 5U105 CFU of C. albicans. In control animals, in¢ltrates are numerous and consist of large amounts of C. albicans both in the yeast form and as pseudohyphae. Only a moderate in£ux of PMNL is found (A). Administration of rG-CSF as late as 24 h after infection resulted in a reduced outgrowth of pseudohyphae as well as an enhanced in£ux of PMNL at the sites of infection, as compared to control animals (B). Periodic acid Schi¡ (PAS) staining; original magni¢cation, 25U.
time points in models of subacute and less overwhelming disseminated candidiasis or when combined with anti-fungal drugs. In the model of rapidly lethal Candida infection, the e¡ect of a single dose of rG-CSF was greatest when given 24 h before injection of C. albicans. When treatment was postponed until 6 h after the onset of infection, the bene¢cial
e¡ect on the survival and organ load of Candida became less pronounced and if administration of rG-CSF was delayed until 24 h after injection of large amounts of C. albicans, a signi¢cant e¡ect on neither mortality nor the outgrowth of microorganisms was found (Fig. 2B) [34]. Nevertheless, such a treatment, even when begun as late as after 24 h, had
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Fig. 4. Survival of mice treated with 500 ng of rG-CSF 6 h after injection of 5U105 CFU of C. albicans, £uconazole (10 mg kg31 bid) or both. Mice (30 animals per group) were injected with a single dose of rG-CSF or saline 1 day before infection. Signi¢cant di¡erence between controls and G-CSF-treated (P 6 0.05) or £uconazole treatment groups (P 6 0.001; Kaplan-Meier log-rank test).
a notable impact on the histopathology at the sites of infection. A signi¢cantly reduced outgrowth of pseudohyphae as well as an enhanced in£ux of PMNs at the sites of infection was seen on microscopic examination in rG-CSF-treated mice, when compared with placebo-treated controls (Fig. 3). When more attenuated or sublethal types of disseminated Candida infection were studied, treatment with rG-CSF for up to 10 days had little e¡ect. The outgrowth of C. albicans in rG-CSF-treated animals did not di¡er signi¢cantly from that in untreated controls (Fig. 2C). This is in agreement with the observations of others, who described a bene¢cial e¡ect on the survival of mice only when rG-CSF was either given in very high doses [35] or in combination therapy with anti-fungal drugs, but only when begun before or immediately after infection and only when small amounts of C. albicans were injected [36,37].
4. Combined therapy with rG-CSF and £uconazole Although the results of treatment of experimental candidiasis with rG-CSF are encouraging, it may be more clinically relevant to determine whether rGCSF can augment anti-fungal drug treatment rather
than stand on its own. As an anti-fungal drug, we selected £uconazole, because it has been shown to be the drug of choice for treatment of candidemia and disseminated infection with C. albicans [3,38]. At 6 h after the onset of experimental disseminated candidiasis in non-neutropenic mice, treatment was started with either rG-CSF, £uconazole or both. In a potentially lethal model of disseminated candidiasis, the mortality was signi¢cantly reduced by treatment with rG-CSF alone, as well as by £uconazole in a dose-dependent fashion (Fig. 4). Although combined therapy appeared not to have an additive e¡ect on survival, the e¡ect of both drugs was additive in inhibiting the fungal burden in the organs of infected mice. Treatment with £uconazole alone led to a signi¢cantly reduced outgrowth of Candida in the kidneys of mice (P 6 0.005), whereas a similar trend was seen in the liver (Fig. 5). The combination of rGCSF and £uconazole signi¢cantly further reduced the numbers of colony forming units (CFU) in both the liver and the kidneys (P 6 0.01) when compared with £uconazole treatment only (Fig. 5). Such an additive e¡ect of rG-CSF and conventional antifungal therapy is in agreement with earlier ¢ndings [35]. Remarkably, whereas the additive e¡ect of rGCSF and £uconazole was limited to reducing the outgrowth of Candida, combined therapy with rG-
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Fig. 5. Outgrowth of C. albicans in the kidneys and liver of mice after i.v. injection of 5U105 CFU of C. albicans. Mice were injected s.c. with 500 ng of rG-CSF or with saline 6 h after the onset of infection, treated with £uconazole (F) at 2.5 or 10 mg kg31 bid or with rGCSF and £uconazole. Each column represents the mean þ S.E.M. for six animals. Signi¢cant di¡erences (P 6 .05) are indicated between control mice and treatment groups (*) and between mice treated with corresponding doses of £uconazole with and without rG-CSF (2, Mann-Whitney U test).
CSF and amphotericin B bene¢cially in£uenced survival of mice [35]. In a model of chronic, non-lethal disseminated candidiasis, prolonged therapy with rG-CSF (500 ng bid) for up to 10 days was investigated, in combination with £uconazole for 3 days. In this model, £uconazole signi¢cantly reduced the outgrowth of Candida CFU during the course of treatment, but rG-CSF did not produce an additional e¡ect (data not shown). From these experiments, it is suggested that rGCSF immunotherapy of experimental disseminated candidiasis in mice is most e¡ective during severe progressive disseminated infections. As was suggested by the paucity of invading PMNs in the infected organs of untreated non-neutropenic mice [34], the local PMN response may be the factor limiting host resistance under those circumstances and exogenous rG-CSF is crucial to in£uence the course of infection.
Although treatment with rG-CSF during less overwhelming models of candidiasis leads to enhanced PMN recruitment and prevention of hyphal outgrowth, the clinical course of infection was not a¡ected. This may indicate that, in subacute infections, PMNs no longer play the pivotal role in host defense, which ultimately determines the outcome of infection. Other mechanisms, such as cell-mediated immunity, are of importance at the later stages of infection. In these types of infection, induction of a Th1-type cellular response, associated with the production of IL-1, TNFK, interferon (IFN)-Q and IL-12, has been show to confer protection against C. albicans infection [21]. The role of PMNs in chronic candidiasis may be primarily as a source of pro-in£ammatory cytokines, since these cells have been shown to produce TNFK, IL-6 and IL-12 upon stimulation with virulent Candida strains, but not non-virulent, agerminative Candida blastospores [39].
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5. Conclusion In conclusion, it has become clear that production of PMNs, their migration to the site of infection, their capacity for killing Candida hyphae and blastospores, as well as their role as producers of pro-in£ammatory cytokines is substantial in the host defense against acute and life-threatening disseminated candidiasis. Experiments in knock-out mice as well as with recombinant cytokines have demonstrated that TNFK, IL-6 and G-CSF are among the mediators that direct the activation and recruitment of PMNs to control invasive Candida infections.
Acknowledgements This work was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy (San Diego, CA, USA), 1998. Part of these studies were supported by an unrestricted grant from Amgen. B.J. Kullberg was supported by a grant from the Royal Netherlands Academy of Arts and Sciences. The experiments were approved by the ethics committee for animal experiments at the Catholic University Nijmegen.
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