Tyrosine kinase activation in response to fungal spores is primarily dependent on endogenous reactive oxygen production in macrophages.

Studies from our laboratory (Shahan, T. A., Sorenson, W. G., and Lewis, D. M. (1994) Environ. Res. 67, 98-104) demonstrated that spores from different fungal species differentially activate rat alveolar macrophages as detected by the measurement of superoxide anion and cytokine production (Shahan, T. A., Siegel, P. D., Sorenson, W. G., Kuschner, W. G., and Lewis, D. M. (1998) Am. J. Respir. Cell Mol. Biol. 18, 435-441). Spores from Aspergillus candidus stimulated production of the highest levels of superoxide anion (5.2 nmol/1.0 x 10(6) alveolar macrophages (AMs)/30 min), followed by those from Aspergillus niger (2.4 nmol/1.0 x 10(6) AMs/30 min) and Eurotium amstelodami (0.4 nmol/1.0 x 10(6) AMs/30 min). The mechanism of this differential activation was studied. Our data demonstrate that the tyrosine kinases p56(Hck), p72(Syk), p77(Btk), p62(Yes), p56(Lck), and p59(Fyn) were specifically activated in response to spores from A. candidus, whereas spores from either A. niger or E. amstelodami activated p56(Hck), p72(Syk), and p77(Btk). Kinetic analysis of specific tyrosine kinases demonstrated that p56(Hck), p72(Syk), and p77(Btk) were activated faster and to a greater extent by spores from A. candidus as compared with spores from E. amstelodami. These data suggest a relationship between reactive oxygen species and tyrosine kinase activation. Treatment of AMs with H(2)O(2) (1 mM) caused the activation of p72(Syk) only, whereas treatment with superoxide dismutase and catalase before treatment with the spores had no effect on tyrosine kinase activation. Incubation with NADPH oxidase inhibitors inhibited both superoxide anion production and the activation of p56(Hck), p72(Syk), and p77(Btk) in response to fungal spores. These data indicate that endogenous reactive oxygen species are necessary for the activation of p56(Hck), p72(Syk), and p77(Btk) by spores; they also indicate that some species of spores are capable of activating tyrosine kinases independent of superoxide anion.

Immune cell types including polymorphonuclear leukocytes (PMNs) 1 and macrophages respond to foreign matter and mi-croorganisms by the production and release of immune mediators, antimicrobial agents, reactive oxygen species (ROS), and proteolytic enzymes (1,2). Superoxide anion O 2 . and its dismutated product, hydrogen peroxide (H 2 O 2 ), are powerful oxidants. ROS are potent antimicrobial agents (3) and, under conditions of high microbial load, cause tissue damage (4). Studies by Sorenson et al. (5) demonstrated that fungal spores from defined species can be readily isolated from materials associated with outbreaks of organic matter-induced lung disease. Other studies showed that spores from Aspergillus candidus cause the production of much higher levels of O 2 . from alveolar macrophages (AMs) than spores from Eurotium amstelodami (6). The same spores were also demonstrated to differentially initiate the production of inflammatory cytokines (7). Since O 2 . is produced immediately in response to fungal spore exposure, we hypothesized that ROS production in response to fungal spores regulates signal transduction pathways that mediate inflammatory processes. Apart from the antimicrobial function of ROS, they also function as intracellular signaling molecules. ROS are small molecules that favor their role in the intracellular signaling mechanism (8,9). Eukaryotic cells possess enzymes capable of rapid and precise regulation of intracellular ROS levels (9). Additional evidence suggesting a role for ROS as signal transduction mediators includes their ability to activate the nuclear transcription factor NF-␤ (10) as well as the demonstration that nitric oxide regulates neurotransmission and cell-mediated immune responses (11).
ROS have been demonstrated to be important in the regulation of protein-tyrosine kinase activation. Schieven et al. (12) showed that treatment of PMNs with H 2 O 2 activates the cytoplasmic tyrosine kinases p56 Lck , p72 Syk , p59 Fyn , and Zap70. In addition, ROS have also been shown to regulate extracellular signal-regulated kinases 1 and 2 in PMNs (13).
ROS are produced through a multicomponent enzyme, NADPH oxidase, which is located in the cell membrane of most leukocytes; however, most cells produce ROS as a side product of electron transport (14). Assembly of the oxidase in response to a stimulus imparts the ability to transfer one electron from NADPH to molecular oxygen to make O 2 . . Dismutation or reaction with other molecules can yield hydrogen peroxide, hydroxyl radical, peroxynitrate, and hypochlorous acid (15).
To investigate the effect of NADPH oxidase and ROS on tyrosine kinase activation, Brumell and Grinstein (16) activated the enzyme by incubating PMNs with guanosine-5Ј-O-(3thiophosphate) and NADPH. Activating the NADPH oxidase in * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. this manner eliminates receptor-operated phosphorylation, which could activate signal transduction pathways other than those directly linked to O 2 . production. Their data showed that p56 Hck , p72 Syk , and p77 Btk were activated, and they concluded that endogenous ROS are able to regulate tyrosine kinase production.
In preliminary studies, we observed that AM treatment with fungal spores from different species stimulated O 2 . production with a concomitant increase in tyrosine phosphorylation of numerous proteins. With the demonstrated control that fungal spores have upon tyrosine phosphorylation, it follows that we would examine the effects of fungal spores and O 2 . on the activation of specific tyrosine kinases in AMs. In this study, we demonstrate that spores from different fungal species differentially activate tyrosine kinases. The phosphorylation and activation of individual tyrosine kinases are demonstrated to be due to the production of endogenous ROS, as evidenced by the inhibition of their activation in the presence of ROS scavengers, and unique molecular determinants on fungal spores. The ultimate goal of this research is to explain the mechanism that regulates the differential cellular responses (ROS production and cytokine production) to fungal spores.
Fungal Spores-Aspergillus niger van Tieghem, A. candidus Link ex Link, and Eurotium amstelodami Mangin (Aspergillus amstelodami Thom et Church) were isolated from dust samples associated with episodes of organic dust-induced chronic pulmonary disease as described previously (5) and were maintained by lyophilization. Spores were killed by autoclaving in endotoxin-free phosphate-buffered saline at 121°C and 15 p.s.i. for 30 min and washed five times with the same buffer before use (6).
Alveolar Macrophage Isolation-Cells were harvested from several male rats (Harlan Sprague-Dawley, Taconic Farms Inc., Germantown, NY) by bronchial alveolar lavage using Ca 2ϩ -and Mg 2ϩ -free Hanks' balanced salt solution, pooled, and washed with the same solution (6). Cell densities were determined by a Coulter counter with a channelizer (Coulter Electronics, Miami Lakes, FL) before plating. AMs comprised 94 -98% of the total bronchial alveolar lavage cell population. Cells were allowed to rest for 1 h before use. Representative samples of bronchial alveolar lavage fluid as well as cell culture medium were tested for endotoxin by the Limulus amebocyte lysate assay (Kinetic QCL, BioWhittaker, Inc., Walkersville, MD).
Measurement of Superoxide Anion-Superoxide anion was measured as described previously (6). AMs (1.0 ϫ 10 6 cells) were incubated with fungal spores (1 ϫ 10 7 ) for 30 min at 37°C or other agents as described below in the presence of cytochrome c. Superoxide anion released from AMs was measured by analysis of cytochrome c reduction detected spectrophotometrically at 550 nm (6). Superoxide anion concentration was determined by measuring the change in absorbance over time in corresponding tubes containing superoxide dismutase. The results are expressed as nanomoles of O 2 . /1 ϫ 10 6 AMs/30 min. The vehicle control consisted of cell medium alone. SDS-PAGE and Immunoblotting-AMs (1 ϫ 10 7 /ml) were used untreated or were incubated with fungal spores (1 ϫ 10 8 ) or other agents as described below for 0 -10 min. Cells were lysed in boiling SDS loading buffer (17) appended with preactivated sodium orthovanadate (0.2 mM) and NaFl (0.3 mM). Samples were separated by electrophoresis through a 12% SDS-polyacrylamide gel and blotted onto polyvinylidene difluoride membranes. Blots of whole cell lysates (WCLs) or immunoprecipitates were probed with the phosphotyrosine-reactive mAb PY20 (1:3500) or with specific tyrosine kinase-reactive antibodies (1:5000) as described above. The blots were then probed with horseradish peroxidase-labeled secondary antibodies (1:5000), developed with enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech), and exposed to x-ray film (Fuji Film, Tokyo, Japan). Some autoradiographs were analyzed with an SI personal densitometer (Molecular Dynamics, Inc., Sunnyvale, CA).
To study the effects of oxidizing agents and antioxidants, AMs were incubated with H 2 O 2 (1 mM) and/or xanthine ϩ xanthine oxidase (described below) for 0 -10 min. Alternatively, AMs were pretreated with superoxide dismutase (1250 units/ml) and/or catalase (500 units/ml) before the addition of oxidants and/or fungal spores. Immunoprecipitates from unstimulated AMs were also incubated with H 2 O 2 (1 mM) for To determine the effect of the NADPH oxidase and the ROS that it produces, cells were treated with the enzyme inhibitor deoxy-D-glucose (125 mM), diphenyleneiodonium (1250 M), or iodonium biphenyl (1250 M) (19) for 45 min prior to the addition of spores. To determine the effect of endogenous ROS production on tyrosine kinase activation, cells were treated with N-acetylcysteine (NAC) (20) antioxidant for 45 min at 37°C before the addition of fungal spores or other agents as described below.
Immune Complex Kinase Assays-AMs (1 ϫ 10 7 cells) were incubated alone or with spores from either A. candidus or E. amstelodami (1 ϫ 10 8 ) for 0 -10 min at 37°C. Cells were lysed in ice-cold lysis buffer composed of 1% Nonidet P-40, 20 mM Tris base (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 1 M aprotinin, 4 M chymostatin, and 20 M leupeptin for 15 min at 37°C. Lysates were centrifuged at 10,000 ϫ g for 10 min to remove insoluble cellular material and then precleared by the addition of 40 l of Sepharose beads alone and incubated for 15 min with gentle mixing on a rotary mixer. Precleared AM lysates were incubated with specific tyrosine kinase-reactive antibodies for 90 min at 4°C with gentle mixing. Immune complexes were immunoprecipitated by the addition of protein A-Sepharose beads, which were previously incubated in a 10% bovine serum albumin solution in lysis buffer, and incubated for 45 min at 4°C. The immunoprecipitates were washed three times and resuspended in kinase assay buffer (7 M MnCl 2 and 20 mM MOPS (pH 7.3)) (21). The kinase activity of the immune complexes was determined essentially as described by Burkhardt and Bolen (22). Briefly, immunoprecipitates were washed with kinase assay buffer; and the autophosphorylation of the different kinases, as well as their ability to phosphorylate the exogenous substrate rabbit muscle enolase, was examined by incubation in kinase assay buffer containing 15.5 Ci of [␥-32 P]ATP and 0.75 M ATP. Enolase determinations indicate the activity level of an individual kinase with respect to its ability to phosphorylate other substrates. Samples were incubated at 27°C on a mixer (slow speed) for 0 -10 min, and the reaction was stopped by the addition of an equal volume of boiling 2ϫ SDS loading buffer. The samples were separated by SDS-PAGE as described, stained with Coomassie Blue, and dried in cellophane. Radioactivity within the dried gels was directly measured with a Molecular Dynamics PhosphorImager using ImageQuant software. Alternatively, dried gels were exposed to x-ray film.
Statistics-All data represent the means Ϯ S.D. of at least three independent experiments with AMs harvested from rats obtained at different times. The blots are representative of data obtained from at least three blots. Statistically significant differences between O 2 . levels were analyzed using a modified multiple inference Student's t test.
Mean differences were considered statistically significant if p values were Ͻ0.05 (23). Fungal Spores from Different Species Differentially Induce Tyrosine Phosphorylation in AMs-Phosphorylation of tyrosine residues has been demonstrated to be an important determinant in the regulation of cellular functions. To examine the effect of fungal spores on protein tyrosine phosphorylation, WCLs from spore-treated AMs were separated by SDS-PAGE and analyzed by Western blotting using the phosphotyrosinereactive mAb PY20. At 5 min post-exposure, the level of phosphotyrosine-containing proteins increased greatly in WCLs from A. candidus (Ac) spore-treated AMs (Fig. 2). In comparison, protein tyrosine phosphorylation levels were lower in response to spores from both A. niger (An) and E. amstelodami (Ea) at 5 min post-exposure (Fig. 2). As a control, latex beads (LB) were incubated with AMs and caused little tyrosine phosphorylation (data not shown). The vehicle control caused no tyrosine phosphorylation. To differentiate between phosphorylated proteins in AMs as compared with fungal spores, spores were incubated alone in lysis buffer, and WCLs were separated as described above. There were no proteins isolated, which indicates that the cell lysis buffer is incapable of lysing these fungal spores (data not shown).
Second, we examined the effect of fungal spores on the phosphorylation of specific tyrosine kinases in AMs following exposure to fungal spores. This was done by immunoprecipitation of the AM WCLs with specific tyrosine kinase-reactive mAbs without prior treatment or following incubation with either A. candidus or E. amstelodami for either 0 or 5 min at 37°C. The immunoprecipitates were separated by SDS-PAGE, followed by Western blot analysis with the phosphotyrosine-reactive mAb PY20. In response to spores from A. candidus, p56 Hck , p72 Syk , p77 Btk , p62 Yes , p56 Lck , and p59 Fyn were activated (Fig. 4A), whereas fungal spores from E. amstelodami activated only p56 Hck , p72 Syk , and p77 Btk (Fig. 4B). The failure of spores from the latter to activate p62 Yes , p56 Lck , and p59 Fyn is potentially interesting. Fig. 4 (A and B) also shows that p53/56 Lyn , p145 Abl , and p59 Fgr were activated at 0 min, which indicates that they are autophosphorylated. This was further confirmed using immune complex kinase assays (data not shown). These data suggest that specific tyrosine kinase activation by fungal FIG. 3. Identification of specific tyrosine kinases present in alveolar macrophages. Rat AMs were collected as described under "Experimental Procedures." AMs (1 ϫ 10 7 cells) were lysed in boiling sample buffer and separated by SDS-PAGE. Proteins were transferred to a PVDF membrane; lanes were cut from the membrane; and each lane was probed with an individual tyrosine kinase-reactive mAb at 1:5000 (p135 Tyk2 , p90 Rsk , p56 Lck , p59 Fyn , p53/56 Lyn , p56 Hck , p62 Yes , Zap70, p145 Abl , p77 Btk , p72 Syk , and p59 Fgr ) and then with a speciesspecific horseradish peroxidase-labeled secondary antibody and detected with ECL. The assay was also performed with rabbit nonimmune serum as a control and failed to detect any proteins. The data are representative samples of experiments performed on three different occasions. FIG. 2. Effect of spores from different fungal species on AM tyrosine phosphorylation. Rat AMs were collected as described under "Experimental Procedures." Cells (1 ϫ 10 7 /ml) were incubated with spores (1 ϫ 10 8 ) from different fungal species for 5 min at 37°C. Cells were then immediately boiled in sample buffer, centrifuged, and separated by SDS-PAGE. Proteins were transferred to a PVDF membrane and probed with a horseradish peroxidase-labeled phosphotyrosinereactive mAb (1:3500), followed by detection with ECL. AMs were exposed to spores from A. candidus (Ac), A. niger (An), and E. amstelodami (Ea); latex beads (LB); or cells alone (not treated (NT)). The data are representative samples of experiments performed on three different occasions.
spores is species-specific.
Kinetic Analysis of Specific Tyrosine Kinase Activation in Response to Spores from Different Fungal Species-Of those tyrosine kinases that were found to be expressed and activated, we specifically studied the kinetic activation of p56 Hck , p72 Syk , and p77 Btk in response to the same fungal spores by immune complex kinase assays alone or in the presence of exogenous rabbit muscle enolase. The kinases p53/56 Lyn , p145 Abl , and p59 Fgr were not studied in depth because they were found to be autophosphorylated (Fig. 4); p62 Yes , p56 Lck , and p59 Fyn were also excluded from further study because they were shown to be activated in response to spores from only A. candidus (Fig.  4). In response to spores from A. candidus, all three kinases were rapidly phosphorylated, first detected at 1 min, and peaked within or at 5 min (Fig. 5A). In response to spores from E. amstelodami, p56 Hck , p72 Syk , and p77 Btk were rapidly phosphorylated, detected at 1 min, and peaked between 5 and 10 min (Fig. 5B), whereas latex beads failed to activate any of these three tyrosine kinases (data not shown). To study the effect of ROS on tyrosine kinase activation, we inhibited the ability of AMs to produce ROS by inhibiting NADPH oxidase with deoxy-D-glucose (125 mM), diphenylenei-odonium (1250 M), or iodonium biphenyl (1250 M) (19), the latter two of which are potent NADPH oxidase inhibitors. Treatment of AMs with these agents inhibited the activation of p56 Hck , p72 Syk , and p77 Btk in response to spores from either A. candidus of E. amstelodami (Fig. 6B). In addition, phosphorylation of p62 Yes , p56 Lck , or p59 Fyn was also unaffected by any of the three agents in response to spores from A. candidus (data not shown). In each case, O 2 . measurements were made using the protocol described under "Experimental Procedures." There was no O 2 . produced by the AMs following treatment with inhibitors or inhibitors and fungal spores. These data indicate that the signal transduction pathway that regulates the activation of these tyrosine kinases in response to fungal spores as described above requires ROS.

Role of ROS in Tyrosine Kinase
To further study the role of ROS as a signal transduction molecule capable of activating tyrosine kinases in response to fungal spores, AMs were incubated with NAC, a powerful antioxidant, before the addition of fungal spores. Treatment of AMs with NAC inhibited the activation of p56 Hck , p72 Syk , and p77 Btk in response to spores from E. amstelodami and A. candidus (Fig. 6B); however, NAC had no effect on the activation of p62 Yes , p56 Lck , or p59 Fyn in response to spores from A. candidus. These data further demonstrate that endogenous ROS are responsible for the activation of p56 Hck , p72 Syk , and p77 Btk , whereas p62 Yes , p56 Lck , and p59 Fyn are activated by a different signaling pathway possibly due to a unique molecular determinant on spores from A. candidus. To determine the effect of ROS scavengers and the NADPH oxidase inhibitor on O 2 . production, AMs were treated with the inhibitors before the addition of fungal spores in the presence of cytochrome c. All three agents inhibited O 2 . production as measured by cytochrome c reduction (data not shown).
It was suggested that ROS may be directly capable of activating these tyrosine kinases. To test this possibility, we isolated individual tyrosine kinases by immunoprecipitation, incubated each with either O 2 . or H 2 O 2 , and looked at the activation of each by Western blot analysis with the PY20 mAb.
Our data indicate that neither O 2 . nor H 2 O 2 was capable of directly activating the kinases p56 Hck , p72 Syk , p77 Btk , p62 Yes , p56 Lck , and p59 Fyn prepared from unstimulated cells (data not shown). Conversely, activated tyrosine kinases from A. candidus spore-treated AMs were unaffected by treatment with NAC or superoxide dismutase. These data indicate that tyrosine kinases are not activated in response to direct interaction with ROS or ROS scavengers. However, treatment of AMs with H 2 O 2 caused the catalase-reversible activation of Syk tyrosine kinase.

DISCUSSION
In this study, we investigated the signal transduction pathways that initiate macrophage activation in response to fungal spores that have been implicated in organic dust-induced lung inflammation. Our data demonstrate that fungal spores from different species differentially activate macrophages. It can be envisioned that the ability of microorganisms to evade immunological detection imparts additional pathogenicity. It is unknown whether these capabilities are due to the absence or presence of a specific ligand-receptor interaction because the immunological determinants on these and most fungi are unknown. Because ROS production is one of the most immediate responses of cell activation and were demonstrated to act as signal transduction mediators in the activation of macrophages, we studied the ability of ROS from fungal spore-treated AMs to influence tyrosine kinase activation.
In this study, we specifically analyzed the mechanism by which fungal spores and ROS produced by AMs in response to these fungal spores lead to tyrosine kinase activation. Our data FIG. 4. Specific tyrosine kinase activation in response to spores from different fungal species. Rat AMs (1 ϫ 10 6 cells) were collected as described under "Experimental Procedures." AMs were incubated with spores (1 ϫ 10 7 ) from A. candidus (A.c; A) or E. amstelodami (E.a; B) for 0 and 5 min. Cells were then lysed, and individual tyrosine kinases were isolated by immunoprecipitation (p56 Lck , p59 Fyn , p53/56 Lyn , p56 Hck , p62 Yes , p145 Abl , p77 Btk , p72 Syk , and p59 Fgr ) with specific mAbs and separated by SDS-PAGE. Proteins were transferred to a PVDF membrane, probed with a horseradish peroxidase-labeled phosphotyrosine-reactive mAb (1:3500), and detected with ECL. The data are representative samples of experiments performed on three different occasions with AMs obtained at different times.
show that spores from E. amstelodami cause the activation of p56 Hck , p72 Syk , and p77 Btk , whereas spores from A. candidus cause the activation of the latter plus p62 Yes , p56 Lck , and p59 Fyn . The significance of these differences is not completely understood. The activation of p56 Hck , p59 Fgr , and p72 Syk by the direct activation of the NADPH oxidase, bypassing cellular receptors, has been previously described by Brumell and Grin-stein (16) and lends additional support to the idea that these kinases are activated through a mechanism involving this enzyme.
Spores from A. candidus stimulated the rapid activation of p56 Hck , p72 Syk , and p77 Btk in AMs. p56 Hck , a member of the Src family of tyrosine kinases, has been shown to be expressed in macrophages and is known to be necessary for tumor necrosis FIG. 5. Kinetic analysis of p56 Hck , p72 Syk , or p77 Btk activation in response to spores from different fungal species. Rat AMs (1 ϫ 10 7 cells) were collected as described under "Experimental Procedures." A, AMs were incubated with fungal spores (1 ϫ 10 7 ) from either A. candidus (Ac) or E. amstelodami (Ea) for 0, 1, 3, 5, 7, or 10 min at 37°C. Cells were then lysed, and individual tyrosine kinases were purified by immunoprecipitation with p56 Hck -, p72 Syk -, or p77 Btk -reactive mAb. Immune complex kinase assays were performed on the immunoprecipitates as described under "Experimental Procedures," separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was exposed to a phosphor screen and x-ray film and analyzed with a PhosphorImager and by autoradiography. factor production in response to lipopolysaccharide (24). The incubation of AMs with either diphenyleneiodonium or NAC decreased tumor necrosis factor production in response to fungal spores and lipopolysaccharide. 2 p72 Syk was also activated after incubation with fungal spores. p72 Syk activation has been shown to be necessary for Fc␥ (CD64)-mediated phagocytosis and is thought to be necessary for immunological responses requiring interleukin-5 (24). p77 Btk , a member of the Tek family, was also demonstrated to be specifically activated in response to these spores. Like the other two kinases, it has been shown to be expressed in neutrophils (25).
Spores from A. candidus alone caused the activation of p62 Yes , p56 Lck , and p59 Fyn . p62 Yes has been shown to be expressed in both monocytes and macrophages (26) and is believed to play a role in cytoskeletal rearrangement because it is commonly found associated with cytoskeletal proteins follow-ing treatment with chemoattractants (27). p56 Lck has also been demonstrated to be expressed in macrophages (28,29). Altogether, the combined research on these tyrosine kinases is limited, so little inference can be made regarding their direct involvement in the differential activation of AMs in response to fungal spores. Indirectly, p56 Hck has been demonstrated to activate the nuclear transcription factor AP-1 in human immunodeficiency virus-infected macrophages (30). Additionally, it has also been shown to activate SP-1, SP-2, and NF-␤ in U937 cells (31). It is for these reasons that we have initiated a study to investigate the ability of fungal spores to activate nuclear transcription factors.
To determine the role of ROS in the mechanism underlying the differential activation of both cells and tyrosine kinases by fungal spores, the activation of tyrosine kinases in response to either H 2 Fig. 5 demonstrates that spores from A. candidus activated tyrosine kinases in AMs more rapidly and to a greater degree than spores from E. amstelodami. These data also indicate that extracellular ROS have little effect on tyrosine kinase activation; however, they indicate nothing about tyrosine kinase activation resulting from receptor-ligand binding activation of NADPH oxidase or endogenous ROS resulting from the latter. The fact that the NADPH oxidase inhibitors deoxy-D-glucose (125 mM), diphenyleneiodonium (1250 M), and iodonium biphenyl (1250 M) inhibit fungal spore-induced O 2 . production as well as tyrosine kinase activation indicates that it is likely that both cell activation and p56 Hck , p72 Syk , and p77 Btk activation in response to fungal spores are regulated through NADPH oxidase. To test this hypothesis, AMs were incubated with NAC, a powerful antioxidant, before the addition of fungal spores to neutralize endogenous ROS. This treatment inhibited the activation of p56 Hck and p77 Btk in response to spores from both species, but not of p62 Yes , p53/56 Lyn , or p59 Fyn in response to spores from A. candidus. Even though the data implicate both the NADPH oxidase and O 2 . in the signaling pathway, the only definitive way to determine the role of NADPH oxidase is to use knockout models or PMNs from humans deficient in or lacking the NADPH oxidase (chronic granulomatous disease of childhood). We are making plans to do so. Because there are likely many other unidentified tyrosine kinases in AMs, neither this study nor any other study surveying specific tyrosine kinases can be absolutely complete. This is the first report to demonstrate a link between cell activation, O 2 . , and specific tyrosine kinase activation in response to fungal spores. More specifically, our data indicate that fungal spores from different species activate tyrosine kinases by endogenous ROS as well as by unique molecular determinants on spores from each fungal species independent of ROS.