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Published online before print July 11, 2007
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* Hematology Section, Department of Oncology, Transplant and Advances in Medicine, University of Pisa, Italy;
Institute of Hematology and Clinical Oncology ‘L. e A. Seràgnoli’, University of Bologna, Italy; and
Department of Infectious Diseases Infection Control and Employee Health and
Transplant Immunology Section, Department of Stem Cell Transplantation and Cellular Therapy, M.D. Anderson Cancer Center, Houston, Texas, USA
1 Correspondence: Department of Oncology, Transplant and Advances in Medicine, Hematology Section, University of Pisa, Ospedale S. Chiara, Via Roma 56, 56100 Pisa, Italy. E-mail: orci{at}sssup.it
ABSTRACT
Aspergillus fumigatus (AF) is a ubiquitous mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. In stem cell transplant recipients, IA now occurs most frequently in the setting of therapy with corticosteroids, including methylprednisolone (MP). We showed previously that gliotoxin (GT), an AF-derived mycotoxin, induces apoptosis in monocytes and dendritic cells, resulting in the suppression of AF-specific T cell responses. We examined the ability of GT to induce apoptosis in polymorphonuclear leukocytes (PMN) and assessed GT effects on important neutrophil functions, including phagocytic function, degranulation, myeloperoxidase activity, and the production of reactive oxygen species (ROS). In contrast to its effects on monocytes, PMN remained resistant to GT-mediated apoptosis. Although many essential neutrophil functions were unaffected, GT inhibited phagocytosis and also induced a decrease in ROS generation by PMN. In contrast, MP therapy potentiated ROS production, suggesting a mechanism that may facilitate tissue injury in IA. Distinct from its effects on untreated PMN, GT augmented ROS production in MP-treated PMN. Our results suggest that although GT may suppress the adaptive immune response, GT may also serve to increase PMN-mediated inflammation, which is likely to play an important role in tissue destruction in the setting of IA.
Key Words: polymorphonuclear leukocytes • micotoxin • immune response
INTRODUCTION
Aspergillus fumigatus (AF) is the most frequent cause of invasive aspergillosis (IA) in immunocompromised patients, including allogeneic stem cell transplant (SCT) recipients [1 2 3 ]. AF is among the most ubiquitous of those fungi capable of producing airborne conidia (spores) and is commonly present in human domiciles. The most predominant manifestation of IA is pulmonary disease [2 ]. Following primary infection, which occurs most commonly through inhalation of conidia, subsequent, further development in the lung may produce a hyphal mass, which may cavitate, with potentially massive destruction of the surrounding lung parenchyma. Following SCT, an initial peak of incidence occurs in the pre-engraftment period associated with neutropenia, and a second peak is often associated with the onset of acute or chronic graft-versus-host disease (GVHD) [4 , 5 ]. Increasingly, IA occurs much more commonly during this latter interval, often in patients with GVHD receiving corticosteroids, including methylprednisolone (MP), at a time when they are no longer neutropenic [6 7 8 9 10 ].
As in the case of other fungi, AF produces a number of mycotoxins, secondary metabolites, which are toxic to humans and animals. Many mycotoxins act as virulence factors by altering host defense systems and by this immunosuppressive activity, help the fungus to invade host tissues [11
]. The most abundant mycotoxin produced by AF is gliotoxin (GT), a hydrophobic metabolite, which belongs to the class of epipolythiodioxopiperazines compounds, characterized by the presence of a quinoid moiety and disulfide bridge across a piperazine ring [5
, 12
]. GT mediates numerous biological activities, nearly all of which are attributed to the oxidized form of the compound containing an intact disulfide bridge. Indeed, structural studies have shown that the biological activities of GT involve the interaction of the polysulfide link with sulfur nucleophiles in a thiol-disulfide exchange [12
, 13
]. GT has been shown to posses a number of immunosuppressive activities [14
]. It can induce apoptosis in various cell lineages [12
, 15
, 16
], including thymocytes [17
], macrophages [18
19
20
], and splenocytes. We showed previously that GT induced relatively specific and rapid apoptosis of monocytes and monocyte-derived dendritic cells (DC), resulting in impaired presentation of target antigens to effector T cells and the attendant limitation of an efficient adaptive immune response [21
]. In addition, GT can affect T cell activity directly, inhibiting the nuclear transcription factor NF-
B, a central regulator of the immune response [22
23
24
25
]. It has also been shown that GT can inhibit the assembly and the function of the NADPH oxidase enzyme complex in human polymorphonuclear leukocytes (PMN), reducing their bactericidal activity [26
27
28
].
Based on our finding that GT preferentially induced apoptosis of monocytes, we hypothesized that similar effects might be seen on marrow-derived PMN, further impairing host responses to AF. To test this hypothesis, we assessed the effects of GT on human PMN, initially examining the ability of GT to induce apoptosis. We assessed the effects of GT further on various functional properties of neutrophils, including phagocytic capability, the ability to release effector granules, myeloperoxidase (MPO) activity, and the production of reactive oxygen species (ROS). Given the important association between MP administration and development of IA, we also examined how the effects of GT on PMN, if any, might interact with the influence of coricosteroids. Overall, our results suggest that GT does suppress ROS production by PMN and impair their phagocytic capacity, although these cells are spared from the apoptosis induction seen in monocytes and DC. It is important that the administration of MP potentiates ROS production by PMN, suggesting a pathogenic model, which may help us to understand the evolution of IA in immunocompromised SCT recipients receiving steroids.
MATERIALS AND METHODS
PBMC and PMN isolation
Healthy donor samples were obtained following informed consent, conforming to principles consistent with those outlined in the Declaration of Helsinki. PBMC were isolated by Ficoll sedimentation using standard protocols. PMN, obtained from healthy donors, were isolated from heparinized venous blood by centrifugation with Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO, USA), and precipitates were obtained as a PMN-rich fraction. Precipitates were then suspended in 1% dextran (T-500, Pharmacia, Piscataway, NJ, USA) and kept for 1 h at room temperature to allow the sedimentation of residual erythrocytes, which were eliminated using a standard lysis buffer (Sigma Chemical Co.). The purity of the PMN obtained was routinely more than 96% by flow cytometry performed using a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA).
Preparation of AF culture filtrate
Water-soluble extracts of AF antigens were prepared using a modification of methods reported by Hebart et al. [29
], as described previously [21
], starting from conidia AF strain AF293, which is used in the AF-sequencing project (Fungal Research Trust, Macclesfield, UK) and was provided by Dr. David Denning (University of Manchester, Manchester, UK).
Assessment of apoptosis using a caspase-3 induction assay
Apoptosis in PMN and PBMC was measured using flow cytometric assessment of activated caspase-3 [21
] (BD Biosciences, San Jose, CA, USA) after a 6-h incubation with or without 35 ng/ml GT (Sigma Chemical Co.), 32 µl water-soluble extracts of AF antigens, MP (100 mM), or MP + GT. PMN and PBMC were permeabilized and stained with antibodies specific for PE-conjugated, active caspase-3 and APC-conjugated CD16 or with PE-conjugated, active caspase-3, PerCP-conjugated CD14, and APC-conjugated CD3. The apoptotic fraction was assessed by flow cytometry as the caspase-3-positive fraction. In PBMC samples, the apoptotic fraction was assessed by flow cytometry by sequential gating on monocytes by scatter and CD14 expression and then on active caspase-3+ cells, and apoptosis in T cells was assessed by sequential gating on lymphocytes by scatter and on CD3+ cells and then active caspase-3+ cells.
Assessment of apoptosis by Annexin V staining
PMN used for assessment of apoptosis by Annexin V staining were isolated as reported previously. The apoptotic percentage in PMN was determined by analyzing phospatidylserine exposure and membrane integrity by double-staining with a FITC-conjugated, anti-Annexin V antibody (BD Biosciences), propidium iodide (PI; Sigma Chemical Co.), and flow cytometric analysis. PMN (106), exposed or not to GT (35 ng/ml or 200 ng/ml) for 1–6 h, were washed with PBS, resuspended in 50 µl Annexin-binding buffer (BD Biosciences), and then incubated for 10 min at room temperature in the dark with a FITC-conjugated, anti-Annexin V antibody and PI (final concentration, 1 µg/ml). The cells were then washed and resuspended in binding buffer, and flow cytometric analysis was performed within 30 min of staining. The extent of apoptosis observed was reported as a percentage using the formula: apoptotic fraction = (AnnexinV+PI–events)/[(AnnexinV+PI–events)+(AnnexinV–PI–events)].
Evaluation of PMN phagocytic activity
Phagocytic activity was calculated as the percentage of phagocytic cells within the PMN population using a NBT test, as reported previously [30
]. PMN were isolated from healthy donors as described previously. Phagocytosis was assessed by resuspending 2 x 106 PMN in 1 ml Hanks' medium and subsequently incubating them for 15 min at 37°C in the following tubes: no stimulation (negative control), PMA (100 ng/ml; Sigma Chemical Co.), or LPS (100 µg/ml; Labogen, Milan, Italy); PMA or LPS + GT (35 ng/ml); and PMA or LPS + GT (200 ng/ml). Subsequently, a 100-µl aliquot of each sample was incubated for an additional 10 min at 37°C with 100 µl NBT solution (Sigma Chemical Co.) suspended at 1 mg/ml in HBSS. After incubation, 40 µl of each sample was used to prepare smears on May-Grumwald-Giemsa-precolored glass slides (Waldeck, Münster, Germany) for microscopic evaluation. PMN were examined visually at 400x magnification, and cells containing dark-blue NBT formazan deposits in the cytoplasm were scored as positive. A single examiner evaluated all slides in a blinded manner to avoid potential bias and maximize consistency.
Flow cytometric evaluation of actin polymerization
Actin polymerization was evaluated by a flow cytometric assay, using FITC-phalloidin (Sigma Chemical Co.) as a specific probe for F-actin [31
]. Isolated PMN were incubated for 20 min at room temperature, with or without GT (35 ng/ml or 200 ng/ml). Samples were stimulated with fMLP at l0–8 M final concentration (Sigma Chemical Co.) in a volume of 1 ml HBSS for 15, 30, 60, and 120 s to evaluate stimulation kinetics. Two control samples were always assessed: one with resting PMN incubated at 4°C and the other with resting PMN incubated at 37°C for 60 s. After incubation and/or stimulation, cells were fixed at 37°C by addition of 3.7% formalin, supplemented with 100 µg lysophosphatidylcholine for 10 min. Thereafter, cells were stained for 25 min at 4°C by adding 5 µl 33 x 10–6 M FITC-phalloidin and then washed and evaluated by flow cytometry within 15 min. Samples were evaluated by a FACScan cytometer (BD Biosciences). Fluorescence gain and photomultiplier tube voltage were evaluated for all samples. Events (105) were acquired for each sample, and results were displayed as histograms. Fluorescence intensity was calculated automatically using CellQuest and was analyzed over a four-decade logarithmic scale. In all instances, the fluorescence histogram yielded a normal distribution. F-actin content in unstimulated PMN and fMLP-induced changes in F-actin content were quantified according to the method described by Wallace et al. [32
]. Relative F-actin content was expressed as the ratio of the test peak channel:control peak channel fluorescence. Two parameters were measured: spontaneous actin polymerization in PMN—ratio of peak channel fluorescence of PMN incubated at 37°C:peak channel fluorescence of PMN stored at 4°C; stimulated actin polymerization—peak channel fluorescence of fMLP test (at each stimulation time):PMN incubated at 37°C.
MPO assay
MPO activity was determined by the oxidation of O-dianisidine dihydrochloride (Sigma Chemical Co.) by H2O2 [6
]. PMN (in HBSS) were activated with LPS (final concentration, 100 µg/ml) and LPS + 35 ng/ml GT for 30 min at 37°C. Unstimulated samples were used as a control. Subsequently, PMN were incubated at room temperature for 15 min with 100 µl O-dianisidine dihydrochloride (1.25 mg/ml in HBSS), supplemented with 0.004% H2O2. The reaction was stopped by the addition of 10 µl NaN3 [1% (wt/vol)]. MPO activity was assessed by measuring the change in absorbance at 450 nm.
Measurement of ROS by flow cytometry
We determined ROS production by a dihydrorhodamine 123 (DHR; Molecular Probes, Eugene, OR, USA) assay and flow cytometric analysis [33
]. Isolated PMN were suspended in PBS at 106 cells/ml. In each instance, we examined nine conditions: a negative control, a positive control (PMA), and seven additional samples incubated with PMA and increasing concentrations of GT (35, 50, 100, 200, 300, 400, or 500 ng/ml). PMN were suspended in 100 µl PBS. Additional PBS (20 µl) was added to the negative control, and 20 µl PMA solution (concentration, 0.01 M) was added to the other sample tubes to stimulate ROS production. Samples were incubated for 15 min at 37°C, and the reactions were terminated by washing treated PMN with ice-cold PBS. PMN were then resuspended in 100 µl PBS and stained with DHR (10 µl of a 10 µg/ml solution) for an additional 10 min at 37°C. After incubation, 500 µl ice-cold PBS was added to the stained cells, which were then kept on ice. The degree of rhodamine fluorescence of stained PMN was analyzed immediately by flow cytometry on at least 105 events. We also assessed ROS production as described in PMN exposed to GT (35 ng/ml), MP (1 mM, 10, mM, 100 mM), or a combination of the two.
Statistical methods
Statistical analyses were performed using Prism statistical software (GraphPad Software, San Diego, CA, USA). Intergroup comparisons were performed using the one-way ANOVA test (for univariate nonparametric group analyses), followed by Tukey's test for analysis of all columns in a pair-wise manner. When appropriate (e.g., when examining the effects of MP), statistical significance was tested using a Wilcoxon matched-pair analysis. All P values were two-tailed and considered significant if less than 0.05. Results were presented using Prism (GraphPad Software) and Illustrator (Adobe, Seattle, WA, USA) software on Macintosh computers (Apple, Cupertino, CA, USA).
RESULTS
GT induces preferential death of monocytes but not PMN
To determine whether PMN were susceptible to GT-mediated apoptosis induction, PMN and control PBMC from six healthy donors were incubated with or without GT (35 ng/ml) or AF crude filtrate (32 µl) for 0, 1, 2, 4, or 6 h. The GT concentration of 35 ng/ml used in this and subsequent experiments was chosen on the basis of our prior findings and as similar or higher concentrations are detectable in the serum of patients with documented or suspected IA [34
]. Using flow cytometry, we assessed apoptosis by quantitating the presence of activated caspase-3 within CD16+ cells (for PMN) relative to that observed within CD3+ and CD14+ subsets (demarcating T cells and monocytes, respectively). We found that neither GT nor the AF filtrate induced significant apoptosis during the interval examined (Fig. 1
). In fact, GT exposure had no influence on the scatter properties of PMN, and the caspase-3-positive fraction was always lower than 2.4% and not significantly different than the control sample (without GT or AF filtrate) at all time-points. In sharp contrast and consistent with our earlier results, the scatter properties of monocytes were changed dramatically following GT or AF filtrate exposure in parallel with an increasing apoptotic fraction, which reached a maximum of 50.1% after 6 h of exposure. Although T cell scatter was unaffected following GT exposure, the apoptotic rate did increase modestly over time, from 0.8% initially to 8.6% at 6 h. Differences in apoptosis induced by GT and the AF filtrate among PMN, monocytes, and T cells were statistically significant (P<0.01) by 2 h of exposure and increased further over time (Fig. 2A
). These results suggest that PMN are resistant to the apoptosis, which is observed in monocytes exposed to GT or AF filtrates [21
].
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Confirmation of absence of PMN susceptibility to GT-mediated apoptosis
We sought to confirm our finding that GT do not induce apoptosis in PMN in an Annexin V assay of apoptosis by flow cytometry. Annexin V positivity represents an early stage in the apoptotic pathway. Isolated PMN were cocultured in the presence of 35 ng/ml or 200 ng/ml GT for 1–6 h. Compared with controls, the percentage of annexin-positive apoptotic cells did not increase during the incubation time with both GT doses (P=NS) and an apoptotic rate never higher than 6% (Fig. 2B)
. These data are matching the results of the caspase-3 evaluation, confirming the lack of an apoptotic effect of GT on PMN.
Effects of GT on PMN phagocytosis
To determine whether GT exerted inhibitory effects on the phagocytic activity of neutrophils, PMN were obtained from seven healthy donors and incubated with or without GT, 35 ng/ml. Phagocytic activity in PMN in the setting of pathogen exposure may be assessed using the NBT test [30
]. The percentage of NBT+ cells within the unstimulated sample (negative control) was 3.3% (SD=3.6), indicative of the background level of activation (Fig. 3
), and activation of PMN with 100 ng/ml PMA (positive control) increased the level of activation to 81.7% (SD=5.0; P<0.01, relative to the control). The concomitant presence of GT resulted in inhibition of PMA-induced activation of phagocytosis in a dose-dependent manner: GT 35 ng/ml: 61.0% (SD=20.9); GT 200 ng/ml: 54.4% (SD=19.1; P<0.05 for GT 35 ng/ml vs. PMA alone and GT 35 ng/ml vs. GT 200 ng/ml; P<0.01 in GT 200 ng/ml vs. PMA). GT alone, 35 ng/ml, or 200 ng/ml did not differ from unstimulated control (P=NS). The use of a more powerful, positive control (LPS, 100 mg/ml) was able to abrogate the dose-dependent, inhibitory effect of GT (data not shown). These results indicated that the phagocytic potential of PMN is inhibited by GT, but this negative effect may be overcome with a maximal activating stimulus.
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AF is an important pathogen in patients who have undergone allogeneic SCT and may cause invasive disease in 4–10% of SCT recipients with an attendant mortality rate ranging from 30% to 80% [1 , 3 , 4 , 35 ]. Previously, we found that GT mediates apoptosis in monocytes, impairing antigen presentation and thereby inhibiting adaptive immune responses by T cells. Given the shared ancestry of monocytes and neutrophils, we reasoned that GT might also exert analogous effects on PMN, thereby impairing another line of host defense important in fungal containment. In this study, we assessed the effects of GT on PMN apoptosis and several other measures of PMN function, including phagocytic capability, degranulation, and ROS production.
Our results indicate that GT, at doses likely to be found in patients with developing IA [13 , 34 ], has important but less dramatic effects on PMN than monocytes. In contrast to its marked, proapoptotic effects on monocytes, which we reported previously [21 ] and confirmed here, we did not observe a significant induction of apoptosis in PMN by GT. In a recently published paper, Bok et al. [36 ] did see induction of PMN apoptosis, using overexpression of the gliZ transcription factor, but we were unable to confirm this finding using purified GT at concentrations, which we found previously in human subjects [34 ] and which we demonstrated to induce marked apoptosis in monocytes and DC [21 ].
Our subsequent experiments suggest GT inhibits, in a dose-dependent manner, the phagocytic capacity of PMN, consistent with the findings of Comera et al. [37 ] published previously. However, we were able to overcome the GT-induced inhibition of PMN phagocytosis in vitro by maximally activating PMN using LPS. In contrast to the inhibitory effects on phagocytosis, PMN exposed to GT are normally able to release granule contents and show an unmodified MPO activity. In addition to the induction of monocyte apoptosis, the inhibition of PMN phagocytosis likely constitutes a separate and important mechanism of immune evasion by Aspergillus species.
In addition to its effects on phagocytic functions, the presence of GT appears to have pleimorphic effects on ROS generation, which are dependent on the presence of glucocorticoids. In the presence of GT alone, even at low but clinically relevant doses, the ROS production of PMN was reduced. As reported previously, GT is able to reduce ROS production [27 ] by inhibiting the assembly of the NADPH oxidase [26 , 28 ]. However, we documented the inhibitory effect of GT at low doses, and enzyme inhibition was reported at much higher GT concentrations, which are not physiologically present in patients with IA [13 , 34 ]. The use of a flow cytometric assay to evaluate ROS production allowed us to document the inhibitory effects of GT even at low concentrations.
It is likely that PMN are the first line of host defense against IA [38 ]. They can remove the inhaled conidia and the hyphal form of AF, preventing tissue invasion [5 , 6 , 39 ]. Our data, which suggest that PMN are resistant to GT-induced apoptosis and that many of their critical functions are unaffected by AF, help to confirm why neutrophils are so critical for the control of AF in vivo [40 ]. In the absence of PMN, or under the circumstance where local GT production impairs phagocytosis, AF can progress rapidly within the lung, leading to invasive disease. The fact that only the hyphal form of AF releases GT suggests that the development of IA depends on a sequence of events, whose net results are an alteration of host defenses resident in lung tissue. These steps include circumvention of the surveillance capacity of PMN, allowing maturation of the hyphal mass, followed by the production of mycotoxins, including GT, which subsequently impair the host cellular immune response [38 39 40 41 ].
Our prior results strongly support the notion that in situ production of GT by AF may play an important role in the pathogenesis of IA. Despite the ubiquitous distribution of AF, only a subset of individuals with compromised immune systems, including SCT recipients, HIV-infected individuals, and patients with chronic granulomatous disease, are known to be at high risk for the development of IA [41 , 42 ]. In addition, as AF is such a ubiquitous, environmental fungus, we thought it likely that significant numbers of AF-specific T cells would be measurable in peripheral blood [29 ]. It is surprising that AF-specific T cells were present at frequencies significantly lower than those of T cells specific for cytomegalovirus, another pathogen seen in a recurring manner by the adaptive immune system [21 ]. Others have reported the difficulty of expanding AF-specific T cells in vivo, which may be the consequence of the paucity of AF-specific T cell precursors in normal individuals [9 , 43 ]. These data, taken together, suggest that although the potential for immune encounters with AF is high, most individuals have frequencies of AF-specific T cells, which are lower than might be expected. Our data are consistent with the notion that in healthy subjects, AF-specific T cells are found at reduced frequencies, as PMN clear conidia efficiently, which are unable to produce GT, from lung tissues, and local GT production, starting with the local hyphal stage, results in APC apoptosis, directly impairing the development of the AF-specific T cell response [42 ].
Despite the importance of PMN in preventing IA development and potentially contributing to the lack of an AF-specific T cell response in healthy subjects, lymphopenia is a significant risk factor for IA during the post-SCT period [9 ]. The rising clinical incidence of IA in the setting of acute or chronic GVHD [5 ] and the high risk of IA in HIV-infected individuals reinforce the importance of the T cell response to AF, as absolute neutropenia is not required for the development of IA in any of these settings. For the obvious reason that corticosteroids impair host T cell responses, the use of MP and other similar agents for therapy of GVHD is associated with an increased risk of IA [6 ]. It is paradoxical that the inflammatory response to AF appears to be exacerbated in the setting of corticosteroid therapy, and high levels of PMN recruitment are evident in pathological specimens in patients with IA. Indeed, a proinflammatory state is often clinically evident in patients with IA when overt clinical progression occurs. Thus, although an inflammatory state may be initiated in an attempt to control a pathogenic infection, its exacerbation may prove detrimental and even contribute to death [6 ].
Our data now suggest that the combination of MP therapy and local production of GT by AF may contribute to inflammation, which may be associated with increased morbidity and mortality as a result of IA. On the basis of pharmacokinetic and pharmacodynamic properties of MP [44 ], we examined the effects of MP in a range of doses (0.5–2 mg/kg/day) typically used in the clinical setting of GVHD therapy. It is interesting that MP was found to increase ROS production paradoxically in combination with GT; in contrast, GT alone reduced ROS production modestly from PMN. Based on the relatively immediate kinetics we observed, it is likely that the effects of MP are not transcriptionally regulated but rather mediated by membrane-bound receptors [45 ]. In fact, after 6 h of exposure, an interval necessary to permit the maximum transcriptional effects of MP [45 ], we saw no additional augmentation in ROS production, consistent with a nontranscriptional regulatory mechanism. As we saw no augmentation in apoptosis by activated caspase-3 analysis, it is also likely that the increase in ROS production was not a direct result of PMN apoptosis [6 , 40 , 46 ].
Our results support a model wherein tissue injury in the setting of IA may occur, not only as a result of the fungus itself but also as a result of the consequences of a PMN-mediated, inflammatory response [6 , 41 ]. In the early, hyphal stage in patients not receiving corticosteroid therapy, AF-derived GT acts to reduce ROS production modestly and to decrease the phagocytic capacity of PMN. Despite the relatively low incidence of IA in patients not receiving corticosteroids, who have normal numbers of circulating PMN, it is likely that this mechanism contributes, at least in part, to evasion of PMN surveillance by AF. However, our current studies also suggest that treatment with MP may not only impair the cellular immune response via well-characterized effects on T cells but also enhance ROS production by PMN responding to AF. These data also suggest that GT potentiates this proinflammatory state further and that the presence of GT and MP results in maximal ROS production. Considered together with our prior findings, the net effect of GT and MP together is an impairment of the AF-specific T cell response as a result of local induction of monocyte and DC apoptosis and an increase of inflammation, which may facilitate increased tissue injury.
In summary, our current results now better define the interactions between AF and host PMN, an essential component of the host immune response against this important pathogen in immunocompromised subjects. These results demonstrate that the effects of AF-derived GT differ significantly in PMN relative to monocytes. In contrast to our prior findings demonstrating potent induction of apoptosis in monocytes and monocyte-derived DC, PMN remain resistant to GT-mediated apoptosis. It is important that the effect of GT in the setting of MP therapy is an increase in ROS production by PMN, suggesting a mechanism that may enhance local tissue injury in the setting of IA. These data suggest that GT has pleiomorphic effects, which may facilitate fungal growth and enhance local tissue destruction in IA. Efforts to improve outcomes related to AF infection in SCT recipients and other immunocompromised patients may be improved by strategies, which may minimize apoptosis induction by GT, reverse the effects of GT on PMN phagocytosis, preserve host T cell responses, and provide alternate GVHD therapies, better sparing host defenses.
ACKNOWLEDGEMENTS
This work was made possible in part by grants provided by "AIL-ONLUS," section of Pisa (to M. P.), by the National Institutes of Health (RO1 CA109328 to K. V. K.), and the Goodwin Family Foundation (to K. V. K.). The authors thank Joyce M. Koenig for the expert technical assistance with ROS determination and for helpful discussions.
Received February 5, 2007; revised June 11, 2007; accepted June 11, 2007.
REFERENCES
B J. Exp. Med. 183,1829-1840
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