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The Journal of Immunology, 2007, 178: 6456-6464.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Perforin-Dependent Cryptococcal Microbicidal Activity in NK Cells Requires PI3K-Dependent ERK1/2 Signaling1

Jeremy C. D. Wiseman*, Ling Ling Ma{dagger}, Kaleb J. Marr*, Gareth J. Jones{ddagger} and Christopher H. Mody2,{dagger},{ddagger}

Departments of * Medical Science, {dagger} Internal Medicine, and {ddagger} Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the {alpha}-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells and CTL are important cellular effectors. They are widely appreciated for their ability to lyse tumor cells or cells infected with a virus. Where CTL function in an adaptive manner, NK cells are considered to be innate effector cells. Despite these differences, both cell populations contribute directly to the protection against viral infection and tumor and mediate the destruction of target cells through two principal mechanisms: Fas-FasL and granule-mediated killing. Another important function of CTL and NK cells is the well-established but less well-known ability to participate in the direct killing of pathogens. Both CTL and NK cells or their products kill a broad range of bacteria, parasites, and fungi (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). In particular, NK cells and CTL mediate direct antifungal activity against the opportunistic fungal pathogen Cryptococcus neoformans (15, 16, 17, 18).

Our previous observations indicate that both NK and CTL are capable of mediating direct anticryptococcal activity (17, 18). Interestingly, CD4 T cell help (or IL-2 or IL-15, which can substitute for T cell help) activates CTL to express granulysin and perforin, whereas NK cells express both molecules constitutively (19, 20, 21). CD8 T lymphocytes are thus capable of killing C. neoformans only after priming and use granulysin but not perforin for effective killing of C. neoformans (17). Importantly, NK cells also posses the ability to directly kill C. neoformans; however, NK cell killing does not require preactivation and employs perforin as the effector molecule (18, 22).

Because C. neoformans is a devastating opportunistic fungal pathogen primarily affecting immunocompromised individuals who lack a functional adaptive immune system, particularly AIDS patients (23, 24), the role of innate cytotoxic effector function is potentially of great importance to the host. In addition to the understanding that perforin is the effector molecule, information about the mechanism of NK cell-mediated killing of C. neoformans is restricted to early studies that describe the binding interaction between NK cells and C. neoformans (15). The binding interaction was observed to require the presence of divalent cations (Mg2+) and induce the polarization of subcellular organelles, including the Golgi apparatus and microtubule organization center, toward the NK-fungal interface. Taken together, this information suggests that the signaling of a granule-mediated killing mechanism is initiated by recognition of the fungus by NK cells; however, despite an appreciation for this important biological function, we do not understand which NK cell receptor system mediates the recognition of C. neoformans, nor is there any further literature, to our knowledge, documenting the signaling of the perforin-mediated killing of C. neoformans or of any other extracellular pathogen. Consequently, all information regarding cytotoxic signaling mechanisms arises from studies of granule-mediated killing in tumor cells.

Signaling of the lytic event during tumor cell recognition and lysis is highly complex and divergent (reviewed in Ref. 25). A large number of activating and costimulatory molecules potentially contribute signals leading to the polarization of the NK cell and lytic granules required for effective killing of the target. Of particular interest is the role of PI3K, which appears to be important under a variety of stimulatory conditions during tumor cell killing (26, 27, 28, 29). Furthermore, during the lysis of tumor targets a specific signaling cascade converging on the activation of PI3K is involved. Sequential activation of PI3K, Ras-related C3 botulinum toxin substrate-1 (Rac-1),3 p21/Cdc42/Rac1-activated kinase (PAK), MEK1/2, and ERK1/2 has been documented to control the redirection of perforin-containing granules. However, not all signaling by NK cells is mediated by PI3K. Previous studies have indicated that signaling can occur in the absence of PI3K catalytic activity; Vav-1 and phospholipase C{gamma}, important for the transduction of Rac-1-dependent signaling and Ca2+ mobilization, respectively, in response to NKG2D ligation can occur in the presence of the specific PI3K inhibitor wortmannin but requires tyrosine phosphorylation of the YINM motif on the cytoplasmic tail of DAP10 (30). Thus, the focus of these studies was to determine whether the fundamentally different microbicidal activity is PI3K dependent or independent and whether the PI3K- ERK1/2 pathway participates in signaling the direct NK cell-mediated killing of C. neoformans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of NK cells and C. neoformans

The YT thymic lymphoma cell line was a gift from Dr. C. Clayberger (Stanford University, Palo Alto CA). YT cells were grown in complete RPMI medium consisting of RPMI 1640, 10% heat inactivated FBS, 0.1 mM nonessential amino acids, 100U/ml penicillin 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine (all from Invitrogen Life Technologies).

Primary NK cells were isolated using a MACS NK cell isolation kit II and the autoMACS automatic magnetic cell sorter, both from Milteny Biotec.

C. neoformans strain CAP67 was obtained from the American Type Culture Collection (ATCC; catalog no. 52817). The fungus was grown on Sabouraud’s dextrose agar (Difco) and maintained on slants, passaged each month as described (31). C. neoformans was grown to log phase in Sabouraud’s dextrose broth (Difco) at 32°C with gentle rotation for 24 h before using it in experiments. For all experiments with primary NK cells the encapsulated C. neoformans strain B3501 was obtained from ATCC (catalog no. 34873) and maintained in the same fashion as C. neoformans CAP67.

Antibodies

All anti-v-akt murine thymoma viral oncogene homologue (c-Akt) and MAPK Abs were obtained from Cell Signaling Technology. The anti-p85{alpha} Ab was part of the PI3K p85{alpha} siRNA/siAb assay kit from Upstate Signaling Solutions and the beta-actin mAb was obtained from Santa Cruz Biotechnology. FITC mouse anti-perforin, mouse anti-perforin, and tetramethyl rhodamine isothiocyanate (TRITC)-goat anti-mouse Abs and isotypes were obtained from BD Biosciences. The secondary Abs IR700 anti-rabbit and IR800 anti-mouse for visualization of immunoblots using the Odyssey infrared imager (LI-COR Biosciences) were obtained from Rockland Immunochemicals. For chemiluminescent detection, anti-rabbit-HRP was obtained from Chemicon International (Temecula, CA) and anti-mouse-HRP was obtained from Cell Signaling Technology.

Immunoblotting

For immunoblot analysis with YT cells or primary NK cells, C. neoformans (CAP 67 or B3501) was added to the culture at an E:T ratio of 1:100 and briefly centrifuged at 27 x g for 5 min to bring them into contract. Cells were lysed in Nonidet P-40 lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1 mM Na3VO4, and 1% Nonidet P-40; all from Sigma-Aldrich). Lysates were sonicated briefly and clarified by centrifugation at 16000 x g for 10 min. Samples were diluted in 50 mM Tris (pH 6.8), 1% SDS, 0.025% bromphenol blue, 10% glycerol, and 20 mM DTT, boiled and resolved by PAGE on NuPAGE Bis-Tris gels (Invitrogen Life Technologies), and then transferred to nitrocellulose or polyvinyldine difluoride membranes (Bio-Rad). Immunoblots for phospho-specific and total Akt and for expression of the p85 subunit of PI3K were visualized using the secondary Abs indicated above and the ECL Plus reagent (Amersham Biosciences). All other immunoblots were visualized using the Odyssey infrared imaging system (LI-COR Biosciences) and secondary Abs according to the manufacturer’s instructions. Densitometry was performed using LI-COR Odyssey instrument software.

Anticryptococcal activity of NK cells

The anticryptococcal activity was determined as previously described by Mody et al. (32). Briefly, C. neoformans (2 x 103/well) were incubated with YT effectors at E:T ratios of (0, 10:1, 200;1, 500:1, and 1000:1) in a 96-well plate. CFU were determined at 0 and 24 h. Data are the means of quadruplicate wells ± SEM expressed as CFU at (t = 24 h) – mean CFU at (t = 0 h), where negative growth indicates killing. For some experiments YT or primary NK cells were pretreated with LY294002 (50 µM), U0126 (50 µM) (Calbiochem), or an equal volume of DMSO as a control. In the small interfering (siRNA) gene-silencing assay, all YT cells were electroporated 72 h before the CFU assay. Viability was assessed for both YT cells and primary NK cells qualitatively by trypan blue exclusion and quantitatively by using a propidium iodide permeability assay, which revealed >90% viability after 24 h of incubation with the inhibitors. Unless otherwise indicated, an E:T ratio of 200:1 was used.

Gene silencing with siRNA

Silencing of PI3K p85{alpha} subunit was performed using the PI3K p85{alpha} siRNA/siAb assay kit (Upstate). To label siRNA for transfection efficiency, the Silencer siRNA labeling kit was used according to the manufacturer’s instructions (Ambion). YT cells were transfected by electroporation. Briefly, YT cells were washed in serum-free medium and resuspended in serum-free medium at (5 x 106/ml). These cells (2 x 106) were added to quartz electroporation cuvettes along with 2.5 µg of FAM-labeled, SMARTpool p85{alpha}-specific siRNA (consisting of four separate 21-nt siRNAs; Upstate), 2.5 µg of nonspecific siRNA (21 nt), or medium alone. A capacitance of 950 and voltage of 275 V was used to electroporate the cells.

NK cell degranulation flow cytometry assay

YT cells were incubated alone or at an E:T ratio of 1:100 with C. neoformans. YT cells were fixed and made permeable using the Cytofix/Cytoperm kit (BD Biosciences). Cells were stained using the FITC-anti-perforin kit (BD Biosciences) and analyzed using a FACScan flow cytometer (BD Biosciences). At least 5000 events were recorded during each analysis.

Immunofluorescence microscopy

For immunofluorescence microscopy, YT cells were mixed together at an E:T ratio of 1:10 with C. neoformans, centrifuged at 27 x g for 5 min, resuspended, transferred to a polylysine-coated, chambered coverglass (Nunc), and incubated for 1 h at (37°C and 5% CO2). Cells were fixed and made permeable using Cytofix/Cytoperm (BD biosciences) and stained with mouse anti-perforin (BD Bioscences) primary Ab and TRITC-goat anti-mouse secondary (BD Biosciences). An isotype-matched Ab was used with secondary TRITC-goat anti-mouse to control for nonspecific labeling. Nuclei were visualized with 4',6'-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). For C. neoformans labeling, 10 x 106 C. neoformans were incubated in 1 ml PBS with 1 mM CFSE (Sigma-Aldrich) for 15 min (37°C and 5% CO2) and the cells were then washed three times in PBS and transferred to complete RPMI medium and incubated 30 min at 4°C, before incubation with YT cells.

Immunofluorescence microscopy was performed on the Olympus IX70 microscope equipped with digital deconvolution (DeltaVision; Applied Precision). Cells were imaged in transmitted light to identify conjugates. Conjugate formation was based on three criteria: 1) YT cells must have been in apparent contact with C. neoformans; 2) demonstrated invagination of cytoplasm; and 3) an elongated morphology. Fluorescence images were then recorded at x60 objective magnification using DAPI/PE-Cy5 and FITC filter sets. The z-sections of fluorescent images were recorded at successive 0.2-µm intervals through the entire thickness of the conjugated YT cells. Image analysis (Softworks software) was used to perform a full iterative or nearest neighbor deconvolution, and images shown in this paper represent a single z-section corresponding to an in-focus NK-fungal synapse.

Statistics

Data expressed are the mean ± the SEM. An ANOVA was performed to establish equal variance and Student’s t test with Bonferroni correction was applied to determine statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NK cell killing of C. neoformans is accompanied by activation of PI3K

To study the signaling of NK cell-mediated anticryptococcal activity, the NK-like cell line YT was used. To determine the effect of YT cells on C. neoformans, the number of viable organisms, as described in previous investigations, was determined (32). Either C. neoformans alone or YT cells mixed with C. neoformans were incubated for 24 h at various E:T ratios (Fig. 1A). These experiments indicated that at the lowest E:T ratio tested (100:1) YT cells mediated a significant (p < 0.0125) inhibition of cryptococcal growth and at higher ratios (≥500:1) the CFU were lower than the starting inoculum, demonstrating cryptococcal killing (Fig. 1A).


Figure 1
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  		FIGURE 1. YT cells kill C. neoformans and activate PI3K following contact with C. neoformans. A, YT cells were incubated at the indicated E:T ratios with C. neoformans at 37°C and 5%CO2 for 24 h. The CFU of C. neoformans alone minus the starting inoculum (C. neoformans) ± SEM was compared with growth in the presence of YT cells minus the starting inoculum at various E:T ratios (from 100:1 to 1000:1). *, p < 0.0125 as compared the growth of C. neoformans alone. The data are representative of three experiments. B, YT cells were left unstimulated (Un) or stimulated with C. neoformans at an E:T ratio of 1:100 for the indicated time period (2–30 min). Phospho-Akt (Ser473) was detected by immunoblot, stripped, and reprobed for pan-Akt. The data are representative of two experiments.

 
To determine whether PI3K signaling pathways were activated during YT cell killing of C. neoformans, YT cells were stimulated with C. neoformans and lysed and immunoblotting was performed for phosphorylated Akt/PKB, which is immediately downstream and dependent upon the activation of PI3K. Akt/PKB phosphorylation was detected within 2 min and returned to unstimulated levels after 30 min of stimulation with C. neoformans (Fig. 1B). These results indicate that YT cell killing of C. neoformans is accompanied by the activation of PI3K.

PI3K is required for anticryptococcal activity

Although it was determined that the stimulation of YT cells with C. neoformans induced activation, we sought to determine whether PI3K activity was required for the anticryptococcal activity. Experiments were performed to determine whether a pharmacologic inhibitor of PI3K, LY294002, affected the activity. LY294002 works by reversibly inhibiting the ability of the p110 subunit of PI3K to catalyze the transfer of phosphate from ATP to its substrate (33). YT cells were pretreated with DMSO (as a control) or LY294002 and were compared with untreated YT cells for their ability to kill C. neoformans. In these experiments the viability of YT cells in the presence of the LY294002 was >90%. A significant (p < 0.007) abrogation of anticryptococcal activity was observed in the presence of the inhibitor LY294002 but not in the presence of vehicle alone (Fig. 2A). Indeed, there was no significant difference between the CFU of C. neoformans alone without YT cells and the CFU of C. neoformans incubated with LY294002-treated YT cells, indicating that the killing mediated by YT cells was abrogated in the absence of PI3K signaling.


Figure 2
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  		FIGURE 2. PI3K is required for the killing of C. neoformans (C. neo). A, YT cells were pretreated with 50 µM LY294002 (LY) for 2 h and the anticryptococcal activity was compared with untreated and vehicle control (DMSO)-treated cells. *, p < 0.007. B, A pool of four distinct 21-bp siRNAs to the p85{alpha} subunit of PI3K was labeled with FAM. Transfection efficiency was determined by flow cytometric analysis of the transfected cells at 24 h after transfection. Thick line, Mock transfected YT cells; thin line, FAM-labeled anti-p85 siRNA. C, Cell lysates from YT cells transfected with siRNA to the p85 subunit of PI3K were separated by SDS-PAGE and immunoblotting was performed for p85{alpha} protein expression and beta-actin (loading control) at 72 h after transfection. Lanes from left to right: Mock transfected, nonspecific siRNA (NS), and p85{alpha}-specific siRNA. D, The anticryptococcal activity of YT cells transfected with anti-p85{alpha}-siRNA was compared with YT cells that has been mock transfected or transfected with nonspecific siRNA at 72 h after transfection. *, p < 0.0125. Data are representative of two experiments.

 
The role of the regulatory subunit of PI3K, p85{alpha}, which is specifically required for the catalytic activity of PI3K, was examined. For this purpose, siRNA was used for targeted knockdown of p85. To determine the transfection efficiency following electroporation, the siRNA was labeled with the fluorophore FAM. Flow cytometric analysis of the fluorescent intensity 24 h after transfection revealed that >98% of cells had taken up the siRNA (Fig. 2B). Immunoblot analysis of the p85{alpha} protein in lysates collected at 72 h after transfection demonstrated a marked reduction in the expression of p85 in cells transfected with specific siRNA compared with those transfected with either the nonspecific siRNA or medium alone (Fig. 2C). Although a small reduction in p85 expression was observed in nonspecific siRNA with respect to mock-transfected cells despite consistent protein expression as assessed by beta-actin (Fig. 2C), this did not affect the ability of YT cells to kill C. neoformans. YT cells transfected with siRNA to p85, nonspecific control siRNA, or medium alone were analyzed for anticryptococcal activity (Fig. 2D). There was a significant (p < 0.0125) reduction in the cryptococcal killing by those cells transfected with p85{alpha}-specific siRNA, but not in the control cells (Fig. 2D). Transfection with p85{alpha}-specific siRNA had no adverse effect on YT cell viability compared with control or mock-transfected cells, where viability under all conditions was >90%. Thus, inhibition of the catalytic activity of PI3K or silencing of the p85{alpha} regulatory subunit indicates that PI3K activity is required for the effective killing of C. neoformans by NK cells.

ERK1/2 are activated and required for killing of C. neoformans by YT cells

MAPK signaling has been reported to participate in signaling during NK-mediated tumor cell lysis (28). To determine whether MAPK, ERK1, and ERK2 were activated in YT cells during direct anticryptococcal activity, YT cells were stimulated with C. neoformans over a time course and immunoblotting for dual tyrosine/threonine phosphorylation of p42/44 MAPK (ERK 1/2) was performed (Fig. 3, A and B). ERK1/2 became transiently phosphorylated in YT cells following contact with C. neoformans (Fig. 3, A and B). Relative phosphorylation, as determined by densitometry of phosphorylated and total ERK1/2, exhibited an activation course similar to that observed for Akt with the maximum phosphorylation at 5 min, returning to baseline within 30 min (Figs. 1B and 3B).


Figure 3
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  		FIGURE 3. ERK1/2 is activated in YT cells following contact with C. neoformans (C. neo) and is required for cytotoxicity. A, YT cells were left unstimulated (Un) or stimulated with C. neoformans at and E:T ratio of 1:100 over the indicated time course (2–30 min). Immunoblotting was performed to detect dual Tyr/Thr phosphorylation of ERK1/2 (p-ERK1/2) and total ERK1/2 (pan-ERK1/2). B, Corresponding densitometry from A in arbitrary units. Data are representative of two experiments. C, YT cells were pretreated for 2 h with 50 µM U0126 and anticryptococcal activity was compared with untreated and vehicle control (DMSO). *, p < 0.007. Data are representative of four experiments.

 
Although like Akt MAP kinase signaling was activated in YT cells following contact with C. neoformans, it was not known whether ERK1/2 activation was required for cryptococcal killing. To address this question, YT cells were pretreated with U0126, a specific inhibitor of MEK1 and MEK2, which are the immediately upstream tyrosine/threonine kinases to ERK1 and ERK2. The ability of treated cells to kill C. neoformans was compared with that of untreated or control (DMSO)-treated YT cells (Fig. 3C). YT cells treated with the MEK1/2 inhibitor U0126 were significantly (p < 0.007) impaired in their ability to kill C. neoformans (Fig. 3C). The viability of YT cells in the presence of U0126 was >90%.

MAP kinase signaling is downstream of PI3K during cryptococcal killing

The specific activation and requirement for both PI3K and ERK1/2 has been confirmed; however, because ERK1/2 may become activated through a number of different signaling pathways (34, 35, 36, 37, 38, 39, 40), it was not known whether PI3K and ERK1/2 were being activated independently to contribute to the killing or were functioning as part of a sequential signaling cascade. To address this question, immunoblot analysis for the activation of both PI3K and ERK1/2 was performed in the presence of specific inhibitors to each enzyme (Fig. 4). YT cells were left untreated, treated with vehicle control (DMSO), or pretreated for 2 h with either LY294002 or U0126. Cells were then stimulated with C. neoformans for 5 min or left unstimulated and lysed after 5 min. Lysates were analyzed by immunoblotting for Akt phosphorylation (Fig. 4, A and B). A relatively constant low level of Akt phosphorylation was observed in the absence of stimulation (Fig. 4, A, odd-numbered lanes, and B, open bars). In the presence of C. neoformans; however, relative phosphorylation was increased and the observed increase was sensitive to PI3K inhibition (LY294002) (Fig. 4A, lane 6) but not to the inhibition of MEK1/2 (U0126) (Fig. 4A, lane 8), indicating that the activation of PI3K was prior to ERK1/2 activation. Conversely, ERK1/2 activation (Fig. 4, C and D) was sensitive to both PI3K inhibition (Fig. 4 c; lane 6) and, as expected, MEK1/2 inhibition (Fig. 4C, lane 8), indicating that ERK1/2 is dependent on and downstream of PI3K.


Figure 4
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  		FIGURE 4. ERK1/2 is activated downstream of PI3K during cytotoxicity against C. neoformans. A and B, PI3K activity is insensitive to ERK1/2 inhibition. YT cells were left unstimulated (white bars) or stimulated (black bars) with C. neoformans for 5 min in the presence of medium (Cells Alone), vehicle control (DMSO), 50 µM LY294002, or 50 µM U0126. Lysates were resolved by SDS-PAGE transferred to nitrocellulose and an immunoblotting was performed to detect phospho-Akt (p-Akt) and total-Akt (pan-Akt) as a control; the immunoblotting (A) and the corresponding densitometry (B) are expressed as relative phosphorylation (phospho/total) in arbitrary units. C and D, Cell lysates were prepared as described above and anti-phospho-ERK1/2 (p-ERK1/2) for ERK1/2 activation and total-ERK (pan-ERK1/2) for loading control were analyzed by immunoblotting (C) and the corresponding densitometry (D). The data from each panel are representative of two experiments.

 
MAPK signaling couples PI3K activation to perforin release during YT cell killing of C. neoformans

The killing of C. neoformans by NK cells is dependent upon the cytolytic effector molecule perforin (18). To determine whether MAP kinase signaling downstream of PI3K activation was controlling the release of perforin during YT cell killing of C. neoformans, a flow cytometric technique was used to analyze the release of perforin in response to the stimulation of YT cells with C. neoformans. YT cells were pretreated with medium, control (DMSO), and U0126 or LY294002 to inhibit PI3K or ERK1/2, respectively. Cells were stimulated with C. neoformans for 4 h. Following incubation, cells were stained with an isotype-matched control FITC-conjugated mAb or a FITC-conjugated anti-perforin mAb and analyzed for perforin content by flow cytometry (Fig. 5). Unstimulated cells demonstrated high levels of perforin expression that which remained over the 4-h incubation period (Fig. 5; unstimulated column). YT cells stimulated with C. neoformans, that were pretreated with medium, or the vehicle control demonstrated a loss of fluorescence (Fig. 5, right column (Stimulated), top two rows (YT Alone and DMSO). YT cells pretreated with inhibitors to PI3K or MEK1/2, however, did not demonstrate a loss of fluorescence (Fig. 5, right column (Stimulated), bottom two rows (LY294002 and U0126). Interestingly the geometric mean fluorescent intensity for YT cells pretreated with LY294002 or U0126 and then stimulated with C. neoformans over 4 h was increased, indicating that they did not release perforin in response to stimulation with C. neoformans when PI3K or ERK1/2 were inhibited.


Figure 5
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  		FIGURE 5. PI3K and ERK1/2 are required for perforin degranulation in response to stimulation with C. neoformans. YT cells with no pretreatment (YT Alone), vehicle control (DMSO), or treated with either 50 µM LY294002 or 50 µM U0126 were left unstimulated or stimulated with C. neoformans, at an E:T ratio of (1:100) for 0 or 4 h. Cells were fixed, made permeable, and stained with isotype (dotted line) or FITC-conjugated anti-perforin mAb at 0 h (thin line) and 4 h (thick line). Data are representative of two experiments.

 
The inability to polarize granules in the absence of functional PI3K-ERK1/2 signaling prevents the release of perforin

Having identified the inability of YT cells to release perforin in response to stimulation with C. neoformans under conditions of PI3K or ERK1/2 inhibition, experiments were performed to determine which part of the lytic machinery was defective. Although both PI3K and ERK1/2 are required for the release of perforin, it was possible that PI3K was responsible for controlling the polarization of the granules toward the NK-cell fungal contact point whereas ERK1/2 is important for calcium signaling and granule release (41). Alternatively, sequential activation of PI3K and ERK1/2 may be required for the polarization of perforin-containing granules, because a PI3K-ERK1/2 cascade is used by NK cells killing tumor targets, which controlled the polarization of lytic granules (28). Consequently, we wondered whether the same pathway was active and was controlling the polarization of the granules during direct microbial killing by YT cells. To address this question, YT cells were incubated with CFSE-labeled C. neoformans for 1 h. Following conjugate formation (see Materials and Methods), YT cell-C. neoformans conjugates were fixed, made permeable, and stained for perforin (Fig. 6). YT cells pretreated with a vehicle control (DMSO) were able to polarize perforin effectively toward the YT-C. neoformans synapse (Fig. 6, DMSO). YT cells pretreated with either the PI3K inhibitor (Fig. 6, LY294002) or the MEK1/2 inhibitor (Fig. 6, U0126) formed conjugates but were unable to polarize perforin toward the synapse, suggesting that the inability to release perforin was rooted in the defective polarization of granules during the lytic event.


Figure 6
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  		FIGURE 6. PI3K and ERK1/2 are required for effective polarization of perforin-containing lytic granules toward the NK-cryptococcal synapse. C. neoformans cells were labeled with CFSE and incubated together with YT cells pretreated with vehicle control (DMSO) or either 50 µM LY294002 or 50 µM U0126. At 1 h the conjugates were fixed and stained on chambered coverglass with a mouse anti-perforin primary Ab, a TRITC-labeled goat-anti-mouse secondary Ab, and DAPI. Bar, 10 µm. Slides were then analyzed by immunofluorescence microscopy. Data are representative of two experiments.

 
Primary NK cells signal via the PI3K-ERK1/2-dependent pathway during cryptococcal killing

Having observed that YT cells signal through a PI3K-ERK1/2 pathway controlling the polarization and release of perforin during cryptococcal killing, we felt that it was important to extend these observations to primary NK cells. Experiments were performed using primary NK cells and the encapsulated C. neoformans var. neoformans strain B3501. The CD16/56 profile of isolated NK cells by magnetic cell sorting indicates highly pure (> 96%) CD56+ NK cells (Fig. 7A). Furthermore, isolated primary NK cells effectively killed C. neoformans in a dose-dependent manner at both 24 and 48 h (Fig. 7B). It was observed that, in a similar fashion to YT cells, primary NK cells responded to intact C. neoformans, demonstrating phosphorylation of Akt that peaked at 5 min and returned to basal levels by 30 min (Fig. 7C, lanes 3–8). Phosphorylation of Akt was not observed when stimulated with the C. neoformans culture supernatant for 5 min (Fig. 7C, lane 2), nor did Akt Abs cross-react with the lysate of C. neoformans in the absence of NK cells (Fig. 7C, lane 1). Furthermore, the anticryptococcal activity of primary NK cells was abrogated in the presence of the PI3K inhibitor LY294002 and the MEK1/2 inhibitor U0126 (Fig. 7D). Thus, the mechanism of activation of primary NK cells using an encapsulated isolate of C. neoformans was the same as that for YT cells.


Figure 7
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  		FIGURE 7. Primary NK cells kill C. neoformans, and signal via PI3K and ERK1/2. A, The CD16/CD56 profile of NK cells isolated from peripheral blood. B, NK cells were mixed with C. neoformans at the indicated E:T ratios (1000:1, 500:1, and 250:1) and CFU values were determined after 24 and 48 h of incubation at 37°C and 5%CO2. C, Immunoblots demonstrating the activation of PI3K by Akt phosphorylation following the stimulation of primary NK cells with C. neoformans at an E:T ratio of 1:100. From left to right the lanes indicate lysates of C. neoformans alone (C), NK cells stimulated for 5 min with C. neoformans culture supernatant (S), NK cells left unstimulated (U), or NK cells stimulated with C. neoformans over the indicated time course (2, 5, 10, 20, and 30 min). D, Inhibition of the killing of C. neoformans using the pharmacologic inhibitors of PI3K and ERK1/2, LY294002 and U0126, respectively. *, p < 0.007. Data are representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In these investigations we have identified the following crucial aspects of signaling activated in NK cells during microbicidal activity: 1) PI3K is activated and essential to YT cell cytotoxic function against the fungal pathogen C. neoformans and both the p110 enzymatic and p85{alpha} regulatory subunits are essential to this function; 2) MAPK signaling, more specifically ERK1/2 signaling, is activated and required for YT cell-mediated microbicidal activity; 3) MAPK activation occurs downstream of PI3K and therefore couples PI3K activity to control the lytic event; 4) the PI3K-ERK1/2 pathway is required for the polarization of perforin-containing granules and the release of perforin following conjugate formation; and 5) PI3K signaling and ERK1/2 signaling are required for the effective killing of C. neoformans by primary NK cells.

For NK cell-mediated tumor cytotoxicity it is becoming well documented that PI3K signaling is an important component of the lytic machinery, participating in both Fc receptor-mediated, natural, and NKG2D/DAP10-mediated cytotoxicity (26, 27, 28, 29, 42, 43). Furthermore PI3K p85 can be recruited by potentially important costimulatory and accessory molecules involved in NK cell-mediated cytotoxicity (44, 45, 46, 47). However, the receptor system involved in microbial killing is entirely unknown and important stimulatory signaling events such as the phosphorylation of Rac-1, the guanine nucleotide exchange factor Vav-1, and phospholipase C{gamma} can occur in the absence of PI3K (33). We therefore performed experiments to determine the activation state of PI3K following stimulation with C. neoformans and the requirement for PI3K catalytic activity for the effective killing of C. neoformans by YT cells. As indicated by Akt phosphorylation, PI3K was specifically activated in response to stimulation with C. neoformans, likely representing a crucial event for the development of cytotoxicity. As pharmacologic inhibition with LY294002 abrogated observed cytotoxicity, it was clear that the effective killing of C. neoformans is dependent upon the catalytic activity of PI3K. Furthermore, previous studies have not distinguished which isoform of the regulatory subunit is required for PI3K activity during cytolytic function (27, 28, 48). As indicated by siRNA-mediated gene silencing of the p85{alpha} regulatory subunit of PI3K, we have demonstrated that in YT cells the presence of the p85{alpha} regulatory subunit is required for the killing of C. neoformans, likely due to the function of p85 in the recruitment of the catalytic p110 subunit and catalytic activity of PI3K. Although, we have not ruled out novel p110-independent functions for the p85{alpha} regulatory subunit, these data confirm that PI3K participates in signaling upon the recognition of C. neoformans and that the presence of p85{alpha} is essential for perforin-mediated killing.

In tumor cell killing, PI3K initiates the activation of a specific signaling cascade, Rac-PAK-MEK-ERK1/2, which has been demonstrated by Jiang and colleagues to be important in directing the polarization of perforin-containing granules (28). ERK1/2 activation has been demonstrated in a number of models to be important for NK cell-mediated cytotoxicity initiated through a variety of receptor systems (34, 35, 41, 49). Thus, we analyzed the role of MAPK signaling in our model. We first demonstrate that ERK1 and ERK2 are specifically and transiently activated in YT cells following contact with C. neoformans, as indicated by immunoblotting for dual tyrosine threonine phosphorylation (36), and are required for killing, as inhibition of the upstream tyrosine/threonine kinases for ERK1/2 (MEK1/2) with U0126 abrogated killing. ERK1/2 activation, however, is observed in a number of diverse signaling pathways in NK cells (37, 38, 39, 40), leading us to question whether ERK1/2 and PI3K were activated independently or whether the PI3K-Rac-1-PAK-MEK1/2-ERK1/2 signaling cascade is conserved in the YT cell killing of C. neoformans.

It was therefore necessary to determine whether ERK1/2 activation was downstream of PI3K and part of a specific PI3K-ERK1/2 cascade, suggesting that a conserved PI3K-ERK1/2 pathway controls both tumor killing and direct anticryptococcal activity. Our results indicate that a PI3K-ERK1/2 pathway is being activated by the stimulation of YT cells with C. neoformans, because immunoblotting for Akt phosphorylation and ERK1/2 phosphorylation in the presence of specific inhibitors of PI3K and MEK1/2 shows that the activation of PI3K is insensitive to ERK1/2 inhibition, but ERK1/2 requires PI3K activity.

Although models of tumor cell killing indicate that ERK1 and ERK2 are important for granule mobilization (28, 34, 35), the precise mechanism by which ERK1/2 contributes to polarization is controversial. In an early report examining spontaneous and Ab-dependent tumor cytotoxicity, ERK 2 activation regulated cytotoxicity through Ca2+-dependent signaling but not through control of the cytoskeleton or regulation of conjugate formation (41). We believe the PI3K-ERK1/2 pathway to be controlling the polarization and release of perforin and confirmed this by using a flow cytometry-based degranulation assay to examine the depletion of perforin in response to stimulation with C. neoformans. Inhibition with either the specific PI3K inhibitor LY294002 or the MEK1/2 inhibitor U0126 prevented the release of perforin from YT cells in response to stimulation with C. neoformans. Furthermore, inhibition of PI3K or ERK1/2 prevented the polarization of perforin toward the NK-fungal contact point, demonstrating the requirement of this pathway for polarization of the required effector molecule.

Although the receptor on NK cells that binds Cryptococcus and triggers killing is unknown, a number of receptors have been identified that are used for the recognition of C. neoformans. These include LFA-1 (CD11a/CD18) as well as the C3 complement receptors, CD35 (CR1), CD11b/CD18 (CR3), and CD11c/CD18 (CR4). Additionally, Cryptococcus or its components bind to the mannose receptor, DC-SIGN, Fc{gamma}R, L-selectin, CD14, TLR-2, and TLR-4 (reviewed in Ref. 50). Of these, LFA-1 (CD11a/CD18) is an important activation receptor for YT cells, which do not kill via Fc{gamma}R (51). Moreover there is evidence that PI3K and Erk1/2 are involved in LFA-1 signaling (52, 53). CD18 on human neutrophils binds cryptococcal glucuronoxylomannan and galactoxylomannan (54), and CD18 is involved in phagocytosis and the production of cytokines by macrophages in response to C. neoformans (55). Finally, anti-CD18 mAb treatment of Cryptococcus-infected mice increased the brain loads, demonstrating the importance of CD18 in optimal host defense (56). Consequently, it may be of interest in future studies to examine the receptor that is used in the recognition of C. neoformans by NK cells.

Importantly, the signaling observed during the YT cell killing of C. neoformans is also observed during the killing of C. neoformans by primary NK cells. Akt phosphorylation was observed over a similar time course when stimulating primary NK cells with the C. neoformans, and the pharmacologic inhibition of both PI3K and ERK1/2 abrogated the killing of the fungus.

We have observed that anti-microbial killing shares a conserved PI3K ERK1/2-dependent signaling pathway used in the lysis of tumor targets. This is of particular interest because NK cells interact with C. neoformans though a novel and unconventional mechanism by which various microvilli penetrate the cryptococcal capsule and contact the cell wall; however, there is no broad region of contact that is observed during NK-tumor conjugation (57). Furthermore, there is no evidence for the formation of a traditional cytolytic NK cell immune synapse, an important aspect of tumor killing, which provides supramolecular organization and costimulatory signals (52, 58). Thus, we have a novel situation by which conserved signaling pathways can be activated in NK cells during the recognition and killing of microbial targets, providing evidence for the existence of a novel NK-microbial cytolytic synapse.

In conclusion, we have established a foothold on the signaling used by NK cells during direct microbial killing and have identified a novel situation by which conserved cytolytic machinery is activated in NK cells by microbial targets.


   Acknowledgments
 
We thank Fusun Turesin for technical assistance with flow cytometry and Dr. Pina Collaruso and the CIHR-funded microscopy core for valuable assistance with immunofluorescence microscopy.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by grants from Canadian Institutes of Health Research (to C.H.M.), the Canadian Foundation for AIDS Research (to C.H.M.), and the Jessie Bowden Lloyd Professorship in Immunology (to C.H.M.). C.H.M. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. Back

2 Address correspondence and reprint requests to Dr. Christopher H. Mody, Room 273, Heritage Medical Research Building, University of Calgary, Calgary, Alberta, Canada. E-mail address: cmody{at}ucalgary.ca Back

3 Abbreviations used in this paper: Rac-1, Ras-related C3 botulinum toxin substrate-1; Akt, v-akt murine thymoma viral oncogene homolog; DAPI, 4',6'-diamidino-2-phenylindole; PAK, p21/Cdc42/Rac1-activated kinase; TRITC, tetramethyl rhodamine isothiocyanate; siRNA, small interfering RNA. Back

Received for publication April 20, 2006. Accepted for publication February 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Canaday, D. H., R. J. Wilkinson, Q. Li, C. V. Harding, R. F. Silver, W. H. Boom. 2001. CD4+ and CD8+ T cells kill intracellular Mycobacterium tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J. Immunol. 167: 2734-2742. [Abstract/Free Full Text]
  2. Ochoa, M. T., S. Stenger, P. A. Sieling, S. Thoma-Uszynski, S. Sabet, S. Cho, A. M. Krensky, M. Rollinghoff, E. Nunes Sarno, A. E. Burdick, T. H. Rea, R. L. Modlin. 2001. T-cell release of granulysin contributes to host defense in leprosy. Nat. Med. 7: 174-179. [Medline]
  3. Ellner, J. J., G. R. Olds, C. W. Lee, M. E. Kleinhenz, K. L. Edmonds. 1982. Destruction of the multicellular parasite Schistosoma mansoni by T lymphocytes. J. Clin. Invest. 70: 369-378. [Medline]
  4. Salata, R. A., J. G. Cox, J. I. Ravdin. 1987. The interaction of human T-lymphocytes and Entamoeba histolytica: killing of virulent amoebae by lectin-dependent lymphocytes. Parasite Immunol. 9: 249-261. [Medline]
  5. Khan, I. A., K. A. Smith, L. H. Kasper. 1988. Induction of antigen-specific parasiticidal cytotoxic T cell splenocytes by a major membrane protein (P30) of Toxoplasma gondii. J. Immunol. 141: 3600-3605. [Abstract]
  6. Khan, I. A., K. A. Smith, L. H. Kasper. 1990. Induction of antigen-specific human cytotoxic T cells by Toxoplasma gondii. J. Clin. Invest 85: 1879-1886. [Medline]
  7. Elloso, M. M., H. C. van der Heyde, J. A. vande Waa, D. D. Manning, W. P. Weidanz. 1994. Inhibition of Plasmodium falciparum in vitro by human {gamma} {delta} T cells. J. Immunol. 153: 1187-1194. [Abstract]
  8. Markham, R. B., J. Goellner, G. B. Pier. 1984. In vitro T cell-mediated killing of Pseudomonas aeruginosa, I: evidence that a lymphokine mediates killing. J. Immunol. 133: 962-968. [Abstract]
  9. Markham, R. B., G. B. Pier, J. J. Goellner, S. B. Mizel. 1985. In vitro T cell-mediated killing of Pseudomonas aeruginosa, 2: the role of macrophages and T cell subsets in T cell killing. J. Immunol. 134: 4112-4117. [Abstract]
  10. Stenger, S., D. A. Hanson, R. Teitelbaum, P. Dewan, K. R. Niazi, C. J. Froelich, T. Ganz, S. Thoma-Uszynski, A. Melian, C. Bogdan, et al 1998. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 282: 121-125. [Abstract/Free Full Text]
  11. Beno, D. W., A. G. Stover, H. L. Mathews. 1995. Growth inhibition of Candida albicans hyphae by CD8+ lymphocytes. J. Immunol. 154: 5273-5281. [Abstract]
  12. Petkus, A. F., L. L. Baum. 1987. Natural killer cell inhibition of young spherules and endospores of Coccidioides immitis. J. Immunol. 139: 3107-3111. [Abstract]
  13. Jimenez, B. E., J. W. Murphy. 1984. In vitro effects of natural killer cells against Paracoccidioides brasiliensis yeast phase. Infect. Immun. 46: 552-558. [Abstract/Free Full Text]
  14. Zhou, P., B. L. Freidag, C. C. Caldwell, R. A. Seder. 2001. Perforin is required for primary immunity to Histoplasma capsulatum. J. Immunol. 166: 1968-1974. [Abstract/Free Full Text]
  15. Hidore, M. R., N. Nabavi, F. Sonleitner, J. W. Murphy. 1991. Murine natural killer cells are fungicidal to Cryptococcus neoformans. Infect. Immun. 59: 1747-1754. [Abstract/Free Full Text]
  16. Nabavi, N., J. W. Murphy. 1985. In vitro binding of natural killer cells to Cryptococcus neoformans targets. Infect. Immun. 50: 50-57. [Abstract/Free Full Text]
  17. Ma, L. L., J. C. L. Spurrell, J. F. Wang, G. G. Neely, S. Epelman, A. M. Krensky, C. H. Mody. 2002. CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15. J. Immunol. 169: 5787-5795. [Abstract/Free Full Text]
  18. Ma, L. L., C. L. Wang, G. G. Neely, S. Epelman, A. M. Krensky, C. H. Mody. 2004. NK cells use perforin rather than granulysin for anticryptococcal activity. J. Immunol. 173: 3357-3365. [Abstract/Free Full Text]
  19. Griffiths, G. M., C. Mueller. 1991. Expression of perforin and granzymes in vivo: potential diagnostic markers for activated cytotoxic cells. Immunol. Today 12: 415-419. [Medline]
  20. Hanson, D. A., A. A. Kaspar, F. R. Poulain, A. M. Krensky. 1999. Biosynthesis of granulysin, a novel cytolytic molecule. Mol. Immunol. 36: 413-422. [Medline]
  21. Pena, S. V., A. M. Krensky. 1997. Granulysin, a new human cytolytic granule-associated protein with possible involvement in cell-mediated cytotoxicity. Semin. Immunol. 9: 117-125. [Medline]
  22. Hidore, M. R., N. Nabavi, C. W. Reynolds, P. A. Henkart, J. W. Murphy. 1990. Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans. J. Leukocyte Biol. 48: 15-26. [Abstract]
  23. Dismukes, W. E.. 1988. Cryptococcal meningitis in patients with AIDS. J. Infect. Dis. 157: 624-628. [Medline]
  24. Coker, R. J.. 1992. Cryptococcal infection in AIDS. Int. J. STD AIDS 3: 168-172. [Medline]
  25. MacFarlane, A. W. t., K. S. Campbell. 2006. Signal transduction in natural killer cells. Curr. Top. Microbiol. Immunol. 298: 23-57. [Medline]
  26. Bonnema, J. D., L. M. Karnitz, R. A. Schoon, R. T. Abraham, P. J. Leibson. 1994. Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity. J. Exp. Med. 180: 1427-1435. [Abstract/Free Full Text]
  27. Cerboni, C., A. Gismondi, G. Palmieri, M. Piccoli, L. Frati, A. Santoni. 1998. CD16-mediated activation of phosphatidylinositol-3 kinase (PI-3K) in human NK cells involves tyrosine phosphorylation of Cbl and its association with Grb2, Shc, pp36 and p85 PI-3K subunit. Eur. J. Immunol. 28: 1005-1015. [Medline]
  28. Jiang, K., B. Zhong, D. L. Gilvary, B. C. Corliss, E. Hong-Geller, S. Wei, J. Y. Djeu. 2000. Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells. Nat. Immunol. 1: 419-425. [Medline]
  29. Wu, J., Y. Song, A. B. Bakker, S. Bauer, T. Spies, L. L. Lanier, J. H. Phillips. 1999. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285: 730-732. [Abstract/Free Full Text]
  30. Billadeau, D. D., J. L. Upshaw, R. A. Schoon, C. J. Dick, P. J. Leibson. 2003. NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway. Nat. Immunol. 4: 557-564. [Medline]
  31. Mody, C. H., G. B. Toews, M. F. Lipscomb. 1988. Cyclosporin-A inhibits the growth of Cryptococcus neoformans in a murine model. Infect. Immun. 56: 7-12. [Abstract/Free Full Text]
  32. Mody, C. H., C. L. Tyler, R. G. Sitrin, C. Jackson, G. B. Toews. 1991. Interferon-{gamma} activates rat alveolar macrophages for anticryptococcal activity. Am. J. Resp. Cell Mol. Biol. 5: 19-26. [Medline]
  33. Vlahos, C. J., W. F. Matter, K. Y. Hui, R. F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269: 5241-5248. [Abstract/Free Full Text]
  34. Wei, S., A. M. Gamero, J. H. Liu, A. A. Daulton, N. I. Valkov, J. A. Trapani, A. C. Larner, M. J. Weber, J. Y. Djeu. 1998. Control of lytic function by mitogen-activated protein kinase/extracellular regulatory kinase 2 (ERK2) in a human natural killer cell line: identification of perforin and granzyme B mobilization by functional ERK2. J. Exp. Med. 187: 1753-1765. [Abstract/Free Full Text]
  35. Wei, S., D. L. Gilvary, B. C. Corliss, S. Sebti, J. Sun, D. B. Straus, P. J. Leibson, J. A. Trapani, A. D. Hamilton, M. J. Weber, J. Y. Djeu. 2000. Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway. J. Immunol. 165: 3811-3819. [Abstract/Free Full Text]
  36. Payne, D. M., A. J. Rossomando, P. Martino, A. K. Erickson, J. H. Her, J. Shabanowitz, D. F. Hunt, M. J. Weber, T. W. Sturgill. 1991. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 10: 885-892. [Medline]
  37. Mainiero, F., A. Gismondi, A. Soriani, M. Cippitelli, G. Palmieri, J. Jacobelli, M. Piccoli, L. Frati, A. Santoni. 1998. Integrin-mediated ras-extracellular regulated kinase (ERK) signaling regulates interferon {gamma} production in human natural killer cells. J. Exp. Med. 188: 1267-1275. [Abstract/Free Full Text]
  38. Yu, T. K., E. G. Caudell, C. Smid, E. A. Grimm. 2000. IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway. J. Immunol. 164: 6244-6251. [Abstract/Free Full Text]
  39. Chuang, S. S., P. R. Kumaresan, P. A. Mathew. 2001. 2B4 (CD244)-mediated activation of cytotoxicity and IFN-{gamma} release in human NK cells involves distinct pathways. J. Immunol. 167: 6210-6216. [Abstract/Free Full Text]
  40. Gellert, G. C., R. P. Kitson, R. H. Goldfarb. 2003. Urokinase-type plasminogen activator receptor crosslinking in an NK cell line increases integrin surface expression by the MAP kinase/ERK 1/2 signaling pathway. J. Cell. Biochem. 89: 279-288. [Medline]
  41. Trotta, R., K. A. Puorro, M. Paroli, L. Azzoni, B. Abebe, L. C. Eisenlohr, B. Perussia. 1998. Dependence of both spontaneous and antibody-dependent, granule exocytosis-mediated NK cell cytotoxicity on extracellular signal-regulated kinases. J. Immunol. 161: 6648-6656. [Abstract/Free Full Text]
  42. Jiang, K., B. Zhong, D. L. Gilvary, B. C. Corliss, E. Vivier, E. Hong-Geller, S. Wei, J. Y. Djeu. 2002. Syk regulation of phosphoinositide 3-kinase-dependent NK cell function. J. Immunol. 168: 3155-3164. [Abstract/Free Full Text]
  43. Spaggiari, G. M., R. Carosio, D. Pende, S. Marcenaro, P. Rivera, M. R. Zocchi, L. Moretta, A. Poggi. 2001. NK cell-mediated lysis of autologous antigen-presenting cells is triggered by the engagement of the phosphatidylinositol 3-kinase upon ligation of the natural cytotoxicity receptors NKp30 and NKp46. Eur. J. Immunol. 31: 1656-1665. [Medline]
  44. Aoukaty, A., R. Tan. 2002. Association of the X-linked lymphoproliferative disease gene product SAP/SH2D1A with 2B4, a natural killer cell-activating molecule, is dependent on phosphoinositide 3-kinase. J. Biol. Chem. 277: 13331-13337. [Abstract/Free Full Text]
  45. Inoue, H., M. Miyaji, A. Kosugi, M. Nagafuku, T. Okazaki, T. Mimori, R. Amakawa, S. Fukuhara, N. Domae, E. T. Bloom, H. Umehara. 2002. Lipid rafts as the signaling scaffold for NK cell activation: tyrosine phosphorylation and association of LAT with phosphatidylinositol 3- kinase and phospholipase C-{gamma} following CD2 stimulation. Eur. J. Immunol. 32: 2188-2198. [Medline]
  46. Teng, J. M., X. R. Liu, G. B. Mills, B. Dupont. 1996. CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C. J. Immunol. 156: 3222-3232. [Abstract]
  47. Umehara, H., J. Y. Huang, T. Kono, F. H. Tabassam, T. Okazaki, E. T. Bloom, N. Domae. 1997. Involvement of protein tyrosine kinase p72syk and phosphatidylinositol 3-kinase in CD2-mediated granular exocytosis in the natural killer cell line, NK3.3. J. Immunol. 159: 1200-1207. [Abstract]
  48. Zhong, B., J. H. Liu, D. L. Gilvary, K. Jiang, M. Kasuga, C. A. Ritchey, J. A. Trapani, S. Wei. 2002. Functional role of phosphatidylinositol 3-kinase in direct tumor lysis by human natural killer cells. Immunobiology 205: 74-94. [Medline]
  49. Milella, M., A. Gismondi, P. Roncaioli, L. Bisogno, G. Palmieri, L. Frati, M. G. Cifone, A. Santoni. 1997. CD16 cross-linking induces both secretory and extracellular signal-regulated kinase (ERK)-dependent cytosolic phospholipase A2 (PLA2) activity in human natural killer cells: involvement of ERK, but not PLA2, in CD16-triggered granule exocytosis. J. Immunol. 158: 3148-3154. [Abstract]
  50. Levitz, S. M.. 2002. Receptor-mediated recognition of Cryptococcus neoformans. Nippon Ishinkin Gakkai Zasshi 43: 133-136. [Medline]
  51. Sugie, K., Y. Minami, T. Kawakami, A. Uchida. 1995. Stimulation of NK-like YT cells via leukocyte function-associated antigen (LFA)-1: possible involvement of LFA-1-associated tyrosine kinase in signal transduction after recognition of NK target cells. J. Immunol. 154: 1691-1698. [Abstract]
  52. Perez, O. D., D. Mitchell, G. C. Jager, G. P. Nolan. 2004. LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56+CD8+ natural killer cells. Blood 104: 1083-1093. [Abstract/Free Full Text]
  53. Ferreira, A. M., H. Isaacs, J. S. Hayflick, K. A. Rogers, M. Sandig. 2006. The p110{delta} isoform of PI3K differentially regulates beta1 and beta2 integrin-mediated monocyte adhesion and spreading and modulates diapedesis. Microcirculation 13: 439-456. [Medline]
  54. Dong, Z. M., J. W. Murphy. 1997. Cryptococcal polysaccharides bind to CD18 on human neutrophils. Infect. Immun. 65: 557-563. [Abstract]
  55. He, W., A. Casadevall, S. C. Lee, D. L. Goldman. 2003. Phagocytic activity and monocyte chemotactic protein expression by pulmonary macrophages in persistent pulmonary cryptococcosis. Infect. Immun. 71: 930-936. [Abstract/Free Full Text]
  56. Kawakami, K., Y. Koguchi, M. H. Qureshi, T. Zhang, Y. Kinjo, S. Yara, K. Uezu, K. Shibuya, S. Naoe, A. Saito. 2002. Anti-CD11 b monoclonal antibody suppresses brain dissemination of Cryptococcus neoformans in mice. Microbiol. Immunol. 46: 181-186. [Medline]
  57. Murphy, J. W., M. R. Hidore, N. Nabavi. 1991. Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans. Infect. Immun. 59: 1476-1488. [Abstract/Free Full Text]
  58. Barber, D. F., M. Faure, E. O. Long. 2004. LFA-1 contributes an early signal for NK cell cytotoxicity. J. Immunol. 173: 3653-3659. [Abstract/Free Full Text]



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