Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M313867200 on February 11, 2004

J. Biol. Chem., Vol. 279, Issue 18, 19113-19121, April 30, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/18/19113    most recent
M313867200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Girotti, M.
Right arrow Articles by Leslie, C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Girotti, M.
Right arrow Articles by Leslie, C. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cytosolic Phospholipase A2 Translocates to Forming Phagosomes during Phagocytosis of Zymosan in Macrophages*

Milena Girotti{ddagger}, John H. Evans§, Danielle Burke{ddagger}, and Christina C. Leslie{ddagger}||

From the {ddagger}Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, the Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80206, and the §Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309

Received for publication, December 18, 2003 , and in revised form, February 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Resident tissue macrophages mediate early innate immune responses to microbial infection. Cytosolic phospholipase A2{alpha} (cPLA2{alpha}) is activated in macrophages during phagocytosis of non-opsonized yeast (zymosan) triggering arachidonic acid release and eicosanoid production. cPLA2{alpha} translocates from cytosol to membrane in response to intracellular calcium concentration ([Ca2+]i) increases. Enhanced green fluorescent protein (EGFP)-cPLA2{alpha} translocated to forming phagosomes, surrounding the zymosan particle by 5 min and completely overlapping with early endosome (Rab5) and plasma membrane (F4/80) markers but only partially overlapping with resident endoplasmic reticulum proteins (GRP78 and cyclooxygenase 2). EGFP-cPLA2{alpha} also localized to membrane ruffles during phagocytosis. Zymosan induced an initial high amplitude calcium transient that preceded particle uptake followed by a low amplitude sustained calcium increase. Both phases were required for optimal phagocytosis. Extracellular calcium chelation prevented only the sustained phase but allowed a limited number of phagocytic events, which were accompanied by translocation of cPLA2{alpha} to the phagosome although [Ca2+]i remained at resting levels. The results demonstrate that cPLA2{alpha} targets the phagosome membrane, which may serve as a source of arachidonic acid for eicosanoid production.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Resident tissue macrophages participate in innate immunity by mediating non-opsonic phagocytosis of invading microorganisms via pattern-recognition receptors (1, 2). Zymosan, a Saccharomyces cerevisiae cell wall particle, has served as a model for recognition of microbes by the innate immune system. Zymosan is composed primarily of the carbohydrate polymers mannan, which binds the mannose receptor, and {beta}-glucan, which binds the recently identified receptor dectin-1 and can also directly bind the complement receptor CR3 (3-7). The {beta}-glucan receptor dectin-1 mediates internalization of zymosan and acts together with Toll-like receptors 2 and 6, which are recruited to the phagosome during zymosan uptake and provide the signals for NF-{kappa}B activation and the production of proinflammatory cytokines (8-10).

Zymosan ingestion also promotes inflammation by inducing arachidonic acid release and production of eicosanoids from the cyclooxygenase (COX)1 and 5-lipooxygenase pathways in macrophages (11, 12). Group IVA, cytosolic PLA2{alpha} (cPLA2{alpha}) mediates zymosan-induced release of arachidonic acid and eicosanoid production in mouse peritoneal macrophages because these responses are attenuated in macrophages isolated from cPLA2{alpha}-/- mice and by the cPLA2{alpha} inhibitor pyrrolidine (13, 14). cPLA2{alpha} is regulated post-translationally by phosphorylation and calcium (15, 16). Several functionally important phosphorylation sites in the catalytic domain of cPLA2{alpha} have been identified (17-20). Calcium regulates cPLA2{alpha} by binding the N-terminal C2 domain and inducing translocation from the cytoplasm to Golgi, endoplasmic reticulum (ER), and nuclear membrane (NM) (21-24). Other enzymes in the eicosanoid cascade, COX1, COX2, and 5-lipoxygenase, also localize to ER and NM (25-27). Calcium-dependent translocation of cPLA2{alpha} to the plasma membrane has not been observed in most cells. However, cPLA2{alpha} localizes to cell-cell junctions in confluent endothelial cells and is transiently recruited to the plasma membrane by NADPH oxidase in granulocytes (28, 29).

Phagocytosis of non-opsonized zymosan transiently increases intracellular calcium [Ca2+]i, which acts synergistically with activation of p42/p44 mitogen-activated protein kinases and phosphorylation of Ser505 for full activation of cPLA2{alpha} in macrophages (30). However, the membranes targeted by cPLA2{alpha} during zymosan phagocytosis have not been identified. It has recently been shown that phagosome formation in macrophages involves the fusion of ER with the plasma membrane (31, 32). Considering that cPLA2{alpha} exhibits calcium-dependent targeting to the ER, the site of COX localization, we investigated the possibility that cPLA2{alpha} and COX may localize to the forming phagosome, thus providing a source of arachidonic acid for PGE2 synthesis at the early stages of microbial ingestion. Using time-lapse microscopy, translocation of GFP-cPLA2{alpha} was monitored in macrophages during active phagocytosis of non-opsonized zymosan. We also quantitated changes in [Ca2+]i during phagocytosis and investigated its role in phagosome formation and cPLA2{alpha} translocation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Ad5 cytomegalovirus-GFP control adenovirus was purchased from Qbiogene (Carlsbad, CA). Zymosan and probenecid were from Sigma. Ionomycin was purchased from Calbiochem (San Diego, CA). Fura Red-AM, 20% Pluronic F-127/Me2SO, zymosan-Texas Red, GSII lectin-Alexa fluor 594, Lyso-Tracker Red DND-99 were from Molecular Probes (Eugene, OR). Hanks' balanced salt solution (HBSS) was purchased from Invitrogen. The cPLA2{alpha} inhibitor pyrrolidine was obtained from M. H. Gelb (University of Washington). Before use, zymosan was boiled in PBS as described previously (33). Both labeled and unlabeled zymosan preparations were sonicated and washed in HBSS containing 25 mM Hepes, pH 7.4 (HHBSS), prior to use. cPLA2{alpha} rabbit polyclonal antiserum was generated as previously described (34). Rabbit polyclonal antibodies against GRP78, COX2, and Rab5 were from Affinity Bioreagents (Golden, CO), Cayman Chemical (Ann Arbor, MI), and Stressgen (Victoria, British Columbia, Canada), respectively. Rat anti-mouse F4/80 monoclonal antibody was from Serotec (Oxford, United Kingdom). Rabbit, rat, or mouse Texas Red-conjugated secondary antibodies were from Jackson Laboratories (Bar Harbor, ME). Prior to use for immunofluorescence, cPLA2{alpha}, COX-2, Rab5, F4/80 antibodies, and rabbit preimmune antiserum were cleared of yeast cross-reacting antibodies by incubation with zymosan particles in PBS at 4 °C overnight. Control experiments showed no appreciable staining of zymosan particles above background levels with these cleared antibodies.

Methods
Cell Culture and Adenoviral Infection—Generation of AdGFP-cPLA2{alpha} and its characterization were previously described (35). Resident mouse peritoneal macrophages were isolated from ICR mice as reported elsewhere (33). For microscopy, cells (2.5 x 105) were plated onto the glass well of Plastek dishes (MatTek Co., Ashland, MA) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 µg/ml streptomycin sulfate, 100 units/ml penicillin G, and 0.29 mg/ml glutamine (incubation medium). After incubation for 2 h, cells were washed 3 times in incubation medium and once in Dulbecco's modified Eagle's medium containing 0.1% human serum albumin, then incubated at 37 °C for 2 h in 150 µl of the same medium containing AdGFP-cPLA2{alpha} at a multiplicity of infection of ~250. Incubation medium (1.5 ml) was then added and the cells were incubated at 37 °C in 7.5% CO2 for 24-26 h. Under these conditions GFP-cPLA2{alpha} was expressed at levels comparable with endogenous cPLA2{alpha} as determined by Western blotting. Macrophages were infected with Ad5 cytomegalovirus-GFP control using the same procedure.

Dual Imaging Digital Microscopy of Live Cells—Macrophages infected with AdGFP-cPLA2{alpha} were washed 3 times in HHBSS. Sonicated zymosan, either unlabeled or Texas Red-conjugated, was added at 10 particles/cell. Cells were imaged in HHBSS at room temperature using an Olympus inverted microscope with a x60, 1.25 NA oil immersion objective, EGFP and Texas Red emission filters (Chroma) in a Sutter filter wheel, a triple band fluorescein isothiocyanate/Cy3/Cy5 dichroic mirror and a TILL Imago CCD camera (TILL Photonics). Excitation light for EGFP (488 nm) and Texas Red (555 nm) was provided by a Polychrome IV monochromator (TILL Photonics). TILLvisION software was used for acquisition and analysis. Calculations of bleach rates for EGFP and of changes in fluorescence of a region of interest at the site of phagocytosis and in the cytoplasm over time were determined as described previously (36).

Phagocytosis Assay and Phagocytic Index—The percent of cells undergoing phagocytosis was calculated by counting the number of cells displaying at least one event of complete zymosan phagocytosis 20 min after addition of the particles and dividing it by the total number of cells per field (typically 20-30 cells). The phagocytic index was calculated by dividing the number of phagosomes by the total number of cells in a field, multiplied by the percent of phagocytosing cells (37).

Synchronized Phagocytosis and Immunofluorescence Microscopy—The protocol for synchronized phagocytosis was adapted from Larsen et al. (38). Briefly, macrophages plated in Plastek dishes and either infected or not with AdGFP-cPLA2{alpha} were washed in cold HHBSS and cooled on ice for 5 min prior to addition of zymosan. After incubation on ice for 15 min, cells were washed once with incubation medium at 37 °C and incubated at 37 °C for the indicated times. Cells were rapidly washed in PBS and fixed in PBS containing 3% paraformaldehyde and 3% sucrose for 15 min. After rinsing in PBS, cells were permeabilized in 0.2% Triton X-100 for 15 min, and incubated in PBS containing 10% fetal bovine serum (block solution) for 1 h. Cells were incubated for 2 h in primary antibody diluted 1:100 in block solution, washed in PBS, and then incubated with the appropriate secondary antibody conjugated to Texas Red in block solution for 1 h. EGFP or Texas Red fluorescence were detected on an Olympus microscope and a TILL Photonic system as described above. For the GS-II lectin double immunofluorescence experiments, cells expressing GFP-cPLA2{alpha} were fixed and permeabilized as described above, and GSII-Alexa 594-conjugated lectin was used at a dilution of 1:800 in HHBSS. Cells were washed extensively in PBS and imaged with an EGFP and a Texas Red (for GSII lectin) filter set.

Calcium Imaging—Macrophages expressing GFP-cPLA2{alpha} were washed in HHBSS containing 2 mM probenecid (complete calcium buffer, CCB), incubated in the dark in CCB containing 5 µM Fura Red-AM, 0.02% Pluronic, and 0.001% Me2SO for 30 min at room temperature, and then washed 3 times and incubated in CCB for 30 min. Imaging was performed on an Olympus microscope using a x40, 1.35 NA oil immersion objective and a Fura Red long pass dichroic mirror and emission filter set (Chroma) for the calcium indicator. Fura Red image pairs illuminated at 425 and 470 nm (to detect the calcium-bound and calcium-free form of the indicator, respectively) and EGFP images illuminated at 488 nm to reveal EGFP-cPLA2{alpha} translocation, were taken at 5-s intervals over a period of 30 min. Ratiometric analysis of the 425 nm/470 nm signals was performed using TILLvisION software. To chelate intracellular calcium, macrophages were incubated simultaneously with 5 µM Fura Red-AM and 10 µM BAPTA-AM for 30 min, washed, and incubated in CCB for 30 min prior to imaging. To chelate extracellular calcium, cells loaded with Fura Red-AM were incubated in CCB containing 20 mM EGTA 10 min before imaging. To deplete intracellular stores, 200 nM thapsigargin was added to Fura Red-AM-loaded cells in CCB during imaging either before or after zymosan addition. [Ca2+]i was determined according to Grynkiewicz et al. (39) following in situ calibration at the end of each experiment to determine the Rmin and Rmax using a value of 140 nM for the Fura Red-AM KD (Molecular Probes).

Measurement of Arachidonic Acid Release and PGE2 Production—Arachidonic acid release was measured as described (13). Briefly, macrophages were plated (1 x 106 cells/well) in 24-well plates and incubated overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.2 µCi of [3H]arachidonic acid/well. After washing, cells were incubated in serum-free Dulbecco's modified Eagle's medium containing 0.1% human serum albumin and stimulated with zymosan. The medium was removed at the times indicated, centrifuged at 500 x g for 5 min, and the amount of radioactivity in the supernatant was determined. Cells were scraped in 0.5 ml of 0.1% Triton X-100 to determine the total cellular radioactivity. PGE2 levels in the culture medium of unlabeled macrophages were determined by enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI).

Online Supplemental Materials—Video 1 shows a resident peritoneal macrophage expressing GFP-cPLA2{alpha} during phagocytosis of non-opsonized zymosan. Video 2 shows transient localization of GFP-cPLA2{alpha} (green) to ruffles in a macrophage during phagocytosis of Texas Red zymosan (red). Video 3 shows a macrophage expressing GFP during phagocytosis of non-opsonized zymosan. Further comments on the data can be found in the online legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ionomycin Induces GFP-cPLA2{alpha} Translocation to Golgi, ER, and NM in Macrophages—We previously reported that stimulus-induced arachidonic acid release can be reconstituted in cPLA2{alpha}-/- mouse lung fibroblasts by overexpression of GFP-cPLA2{alpha} using recombinant adenovirus (AdGFP-cPLA2{alpha}) (35). GFP-cPLA2{alpha} also rescued zymosan-stimulated arachidonic acid release in peritoneal macrophages isolated from cPLA2{alpha}-/- mice (data not shown). We first determined the localization of GFP-cPLA2{alpha} in macrophages stimulated with ionomycin. GFP-cPLA2{alpha} rapidly translocated from a predominantly cytoplasmic location to a juxtanuclear region (Fig. 1A), which co-stained with GS-II lectin, a Golgi marker (Fig. 1B). GFP-cPLA2{alpha} fluorescence was also observed in a perinuclear location consistent with ER and NM (Fig. 1B, GFP-cPLA2{alpha}), similar to previous findings that GFP-cPLA2{alpha} translocates to Golgi, ER, and NM in response to long duration calcium transients (24). These data confirm that GFP-cPLA2{alpha} is functional and can be employed to investigate translocation in living macrophages.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1.
Effect of ionomycin on GFP-cPLA2{alpha} localization in macrophages. Macrophages expressing GFP-cPLA2{alpha} were (A) imaged using time-lapse microscopy at the moment of ionomycin (500 nM) addition (0s), and at 5 and 30 s thereafter, or (B) treated with 500 nM ionomycin for 5 min, fixed in paraformaldehyde prior to labeling with GSII-Alexa Fluor 594 lectin, and then imaged using an EGFP filter and Texas Red filter (for GSII-Alexa Fluor 594 fluorescence). Scale bar = 10 µm.

 
GFP-cPLA2{alpha} Translocates to the Phagosome during Zymosan Phagocytosis—The kinetics of GFP-cPLA2{alpha} translocation to membranes was investigated in macrophages during phagocytosis of non-opsonized zymosan by time lapse digital microscopy. As shown in Fig. 2A (see also video 1 in Supplementary Materials), GFP-cPLA2{alpha} translocated to the site of phagocytosis very early on during phagosome formation and preceded phago-lysosome fusion (Fig. 2B). Moreover, GFP-cPLA2{alpha} remained localized to the phagosome for an extended time (40-60 min) after particle internalization (Fig. 2A). On average, the time taken between the first frame where a particle in proximity of a cell was found not to move (considered an indication of binding to the cell surface) and the first frame where cPLA2{alpha} was observed surrounding about 50% of the zymosan particle was 3-6 min. In addition, GFP-cPLA2{alpha} localized to a juxtanuclear region that was reminiscent of Golgi (Fig. 2A). The localization of GFP-cPLA2{alpha} was also detected in ruffles near the site of particle engulfment and on ruffles in areas not directly engaged in zymosan uptake (Fig. 2, C and D; see also videos 1 and 2 in Supplementary Materials).



View larger version (54K):
[in this window]
[in a new window]
  		FIG. 2.
GFP-cPLA2{alpha} translocates to the phagosome. A, GFP-cPLA2{alpha} localization was monitored during zymosan phagocytosis using time-lapse microscopy and an EGFP filter; images are shown at the time of zymosan addition (0m), or indicated times thereafter. A Nomarski image at 66 min (N) shows the position of the internalized zymosan particles (see also video 1 under Supplementary Materials). B, following incubation of GFP-cPLA2{alpha}-expressing macrophages with 50 nM Lyso-tracker Red for 1 h at 37 °C, zymosan phagocytosis was monitored by time-lapse microscopy using a EGFP filter (GFP-cPLA2{alpha}, green) or a Texas Red filter (Lyso-tracker, red); images are shown at the time of zymosan addition (0m) or the indicated times thereafter. C and D, on several occasions, GFP-cPLA2{alpha} fluorescence was transiently detected in areas close to the site of phagocytosis reminiscent of plasma membrane ruffles (indicated by the arrow, C and D are cells from separate experiments; see also video 2 under Supplementary Materials). Scale bar = 10 µm.

 
The increase in fluorescence intensity at the phagosome correlated with a decrease in cytoplasm fluorescence of GFP-cPLA2{alpha} (Fig. 3A). In macrophages infected with AdGFP control virus, fluorescence surrounding the phagosome was not observed nor was there a decrease in cytoplasm fluorescence (Fig. 3B; see also video 3 in Supplementary Materials. The bright dots in these images are due to autofluorescence of zymosan particles.) No localization to phagosomes, Golgi, or ruffles was detected in control cells expressing GFP alone. This type of analysis has been used to discriminate between true and pseudo-localization in cells expressing a GFP-tagged protein versus cells expressing GFP alone (40). Because fluorescence decreased in the cytoplasm as GFP-cPLA2{alpha} fluorescence increased around zymosan particles, it suggests that GFP-cPLA2{alpha} undergoes a true translocation to phagosomes during early phagocytosis.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 3.
Analysis of GFP-cPLA2{alpha} translocation from the cytoplasm. Kinetic analyses of GFP fluorescence of an area of interest in the cytoplasm of a representative cell undergoing phagocytosis and either expressing GFP-cPLA2{alpha} (A) or GFP alone (B) are shown (see also video 3 under Supplementary Materials). The values of fluorescence intensity were background- and bleach-corrected. Data are representative of 5 analyses from three independent experiments. In B (inset) high intensity fluorescent dots represent zymosan autofluorescence.

 
Arachidonic acid release was measured as an indicator of endogenous cPLA2{alpha} activity under the conditions of the microscopy. Zymosan stimulated arachidonic acid release from macrophages incubated either at 37 °C or room temperature, albeit the amount of arachidonic acid released was about 50% less at room temperature (not shown). This confirmed that, under our experimental conditions for microscopy (room temperature), macrophages responded to zymosan with increased cPLA2{alpha} activation and arachidonic acid release. We have previously reported that the cPLA2{alpha} inhibitor pyrrolidine blocks zymosan-induced arachidonic acid release in macrophages. The effect of pyrrolidine on phagocytosis of zymosan was investigated. The percentage of phagocytic cells and the phagocytic index of macrophages treated with 1 µM pyrrolidine (74.5%; 204, respectively) was similar to untreated macrophages (73.5%; 210).

Immunocytochemistry on fixed macrophages not infected with AdGFP-cPLA2{alpha} was also carried out to confirm that endogenous cPLA2{alpha} exhibited similar localization as GFP-cPLA2{alpha}. As shown in Fig. 4, endogenous cPLA2{alpha} localized to forming phagosomes 5 min after the start of phagocytosis of zymosan (A and C) and was in close proximity 60 min later (G). No fluorescence was evident around the phagosome when non-immune rabbit serum was used (E and I). Thus, the GFP-conjugated construct mimicked the endogenous enzyme.



View larger version (125K):
[in this window]
[in a new window]
  		FIG. 4.
Endogenous cPLA2{alpha} translocates to the phagosome. Synchronized phagocytosis of zymosan was performed as described under "Experimental Procedures." Following zymosan internalization for 5 (A-F) or 60 min (G-J), cells were fixed and immunolabeled with an antiserum against cPLA2{alpha} (A, C, and G) or with rabbit non-immune serum (E and I). B, D, F, H, and J are the Nomarski images corresponding, respectively, to the fluorescent images A, C, E, G, and I, to highlight the position of zymosan particles.

 
Dual Label Immunofluorescence of cPLA2{alpha} with Plasma Membrane, Endosomal, and ER Markers—F4/80, a cell surface macrophage protein highly expressed on resident peritoneal macrophages, was used as a marker to monitor plasma membrane internalization during uptake of zymosan (41, 42). F4/80 fluorescence was found around the zymosan particle and on extensions of the plasma membrane adjacent to the forming phagosome 5 min after initiation of phagocytosis, and its localization overlapped entirely with GFP-cPLA2{alpha} (Fig. 5, D, E, and F). Interestingly, while in unstimulated cells F4/80 was distributed along the cell surface (Fig. 5B), in phagocytic cells F4/80 fluorescence was concentrated around the phagosomes with little evidence of plasma membrane stain (Fig. 5E, see also Supplementary Materials Fig. 1), suggesting that F4/80 may also be recruited to phagosomes. The specificity of phagosome localization was confirmed in control experiments that showed the lack of labeling of isolated zymosan particles with the F4/80 monoclonal antibody and with secondary antibody alone (data not shown).



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 5.
Localization of GFP-cPLA2{alpha}, F4/80, and Rab5 in macrophages during zymosan phagocytosis. GFP-cPLA2{alpha} expressing macrophages either unstimulated (A, B, G, and H) or undergoing synchronized zymosan phagocytosis for 5 min (C-F and I-L) were fixed and labeled with antibodies to F4/80 (A-F) or Rab5 (G-L), followed by a Texas Red-conjugated secondary antibody. GFP-cPLA2{alpha} green fluorescence (A, D, G, and J) was detected with an EGFP filter, and a Texas Red filter was used to detect F4/80 (B and E) or Rab5 red fluorescence (H and K). The Nomarski images show the positions of internalized zymosan particles (C and I). Magnifications of the phagosome areas from the merged image are shown (F and L). Scale bars = 10 µm.

 
During their formation, phagosomes interact with and sequentially acquire markers of early endosomes, late endosomes, and lysosomes (43, 44). Rab5 is an early endosome marker that associates with phagosomes within the first 10 min of phagocytosis (45, 46). Immunostaining of GFP-cPLA2{alpha}-expressing macrophages fixed 5 min after initiation of zymosan phagocytosis revealed a co-localization of Rab5 and GFP-cPLA2{alpha} on nascent phagosomes (Fig. 5, J, K, and L).

To determine whether cPLA2{alpha} translocation to the forming phagosome coincided with ER-phagosome fusion, we performed immunocytochemistry on fixed GFP-cPLA2{alpha}-expressing macrophages 5 min after the start of synchronized zymosan phagocytosis, counterstaining with an antibody against the ER resident protein GRP78. As shown in Fig. 6, the ER marker was found in the vicinity of phagocytic cups 5 min after particle addition (C, G, and J). In contrast, cPLA2{alpha} was found to encircle completely the newly engulfed particle (B, F, and I) and overlapped with the ER marker only in the region of the phagocytic cup. Note that images F-H show a basal plane of the cell; I-K show an apical plane of the same cell.



View larger version (50K):
[in this window]
[in a new window]
  		FIG. 6.
Localization of GFP-cPLA2{alpha} and GRP78 during zymosan phagocytosis. Following synchronized zymosan phagocytosis for 5 min, GFP-cPLA2{alpha} expressing macrophages were fixed and stained with antibodies to GRP78, followed by a Texas Red-conjugated secondary antibody. A-D and E-K are images of representative cells from different experiments. GFP-cPLA2{alpha} green fluorescence (B, F, and I) was detected with an EGFP filter, and GRP78 red fluorescence (C, G, and J) was captured with a Texas Red filter. A magnified portion of the phagosome area of the merged image is shown (D, H, and K). Images E-K show a magnification of the phagosomal area of the cell taken either at a basal plane (F-H) or at an apical plane (I-K) of the cell. The Nomarski images (A and E) show the position of the internalized zymosan particle. Scale bars = 10 µm.

 
Zymosan-induced Production of PGE2 and Localization of COX2—Stimulation of mouse peritoneal macrophages with zymosan triggers cPLA2-dependent production of PGE2 (13). As shown in Fig. 7, PGE2 production was greater than control levels 5 min after adding zymosan (inset), and accumulated at a constant rate up to 30 min after zymosan addition. PGE2 levels were attenuated by the COX2 inhibitor NS-398. The presence of COX2 in the mouse peritoneal macrophages was confirmed by Western blotting (data not shown). The localization of COX2, a resident ER protein, was determined by immunofluorescence 5 min after zymosan addition in fixed macrophages expressing GFP-cPLA2{alpha} (Fig. 8, A-H). Similar to our findings with GRP78, COX2 localized in the region of the phagocytic cup, whereas GFP-cPLA2{alpha} completely surrounded the zymosan particle (as shown in two different cells, A-D and E-H). At 15 min after internalization (Fig. 8, J-L), COX2 was distributed more uniformly around the phagosome where it extensively overlapped with GFP-cPLA2{alpha} fluorescence (Fig. 8L).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 7.
Time course of PGE2 production. PGE2 production was measured at the indicated times during internalization of zymosan (squares) and compared with unstimulated cells (diamonds), and to cells preincubated with NS-398 and exposed to zymosan for 60 min (triangle). The inset is a scale-up of PGE2 production at 5 min. Data are the averages ± S.E. of three independent experiments performed in duplicate.

 



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 8.
Localization of GFP-cPLA2{alpha} and COX2 during zymosan phagocytosis. Following synchronized zymosan phagocytosis for 5 (A-H) or 15 min (I-L), GFP-cPLA2{alpha} expressing macrophages were fixed and immunolabeled with antibodies to COX2, followed by a Texas Red-conjugated secondary antibody. GFP-cPLA2{alpha} green fluorescence (B, F, and J) and COX2 red fluorescence (C, G, and K) were detected with EGFP and Texas Red filters, respectively. A magnified portion of the phagosome area from the merged image is shown in D. An overlay of the GFP-cPLA2{alpha} and COX2 fluorescence of another cell is shown in H. Nomarski images (A, E, and I) show the positions of internalized zymosan particles. Scale bars = 10 µm.

 
Calcium Transients during Zymosan Phagocytosis—Previous work established the role of the C2 domain and intracellular calcium requirements for cPLA2{alpha} translocation to ER, NM, and Golgi membranes (21, 24). We investigated the effect of zymosan on changes in [Ca2+]i in relationship to the kinetics of GFP-cPLA2{alpha} translocation to phagosomes. The use of intracellular calcium indicators excitable by near UV wavelengths (such as Fura-2) was not possible, as macrophages displayed a marked sensitivity to prolonged illumination at these wavelengths. This imposed the use of a red-shifted calcium indicator (Fura Red), which allowed concomitant acquisition of green fluorescence but prevented the use of Texas Red-labeled zymosan particles. For this reason we were not able to precisely differentiate between the moment of particle binding and initiation of phagocytosis in these studies. However, phagocytosis and complete internalization of particles was easily detected by inspecting the zymosan autofluorescence signals in overexposed images of the cells.

Addition of zymosan particles induced a biphasic pattern of calcium mobilization in macrophages incubated in CCB (1.3 mM Ca2+) (Fig. 9A). An initial, high amplitude calcium transient was followed by a lower amplitude, sustained phase of [Ca2+]i increase, which displayed random oscillatory behavior. The initial transient occurred on average 14 s after addition of zymosan and lasted 45 s (n = 41, five independent experiments). GFP-cPLA2{alpha} translocation to phagosomes occurred on average 3-6 min after zymosan addition (see Fig. 2A). The rapid calcium response following particle addition was not due to a soluble component of the zymosan preparation because the particles were washed and resuspended in HHBSS immediately before use, and addition of the supernatant from the washing step (storage medium) failed to induce any calcium change. Therefore, the results indicate that the initial transient is the result of the first particle binding to the cells.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 9.
Calcium transients during phagocytosis of non-opsonized zymosan. GFP-cPLA2{alpha} expressing macrophages were loaded with Fura Red-AM and calcium changes were monitored in single cells by time-lapse microscopy, and expressed as ratios of the calcium-bound form (425 nm) and calcium-free form (470 nm) of the indicator after background correction. Calcium changes during phagocytosis of zymosan were monitored in CCB (A, D, and E), or in CCB containing either BAPTA-AM (B) or EGTA (C). The time of zymosan (z) addition is indicated by an arrow. Thapsigargin (TG) is added 10 min after (D) or 3 min before (E) zymosan addition. The inset in C shows a cell undergoing limited phagocytosis in the presence of EGTA and captured at the time indicated by the downward arrow; the calcium trace for this cell is colored in red. In A the trace of a representative cell is shown, and in B-E the traces of 4 cells are shown. The data are representative of 40-60 determinations from three to six independent experiments. For the conditions shown in A-C and E, the number of phagocytic cells (dark blue bars) and the phagocytic index (light blue bars) were determined (F) and expressed as percent of control values.

 
At the peak of the first transient [Ca2+]i was 500-750 nM above the resting levels (30-50 nM). In the oscillatory phase, [Ca2+]i was considerably reduced but was always above prestimulation levels. In GFP-cPLA2{alpha} expressing macrophages the difference between post-zymosan and pre-zymosan [Ca2+]i levels ({Delta}[Ca2+]i) in the sustained phase was on average 130 ± 40 nM (n = 13, three independent experiments). These values were very similar to those observed in zymosan-stimulated, uninfected macrophages (average {Delta}[Ca2+]i = 104 ± 56 nM, five determinations). The number of peaks in the oscillatory phase did not consistently coincide with uptake of zymosan particles. Moreover, some cells undergoing phagocytosis did not display clear oscillations in this phase, at least at the temporal resolution of our experiments, but they did show elevated [Ca2+]i after the initial calcium transient.

Chelation of intracellular calcium with BAPTA-AM abolished both phases of calcium increase (Fig. 9B). Chelation of extracellular calcium with EGTA did not affect the timing, duration, and amplitude of the first transient but it abolished the sustained [Ca2+]i increase and the oscillations (Fig. 9C). This indicates that the secondary phase of [Ca2+]i increase, but not the first transient, depends on influx of extracellular calcium. Addition of the SERCA pump inhibitor thapsigargin to macrophages during the low amplitude, sustained calcium increase after zymosan addition resulted in complete elimination of the oscillatory trends in all cells analyzed (Fig. 9D), but did not affect the elevated [Ca2+]i in the sustained phase. This indicates that the oscillations require functional intracellular calcium stores. Addition of thapsigargin to macrophages before addition of zymosan prevented the first transient elicited by zymosan (Fig. 9E), indicating that it is the result of release of calcium from intracellular stores. This is consistent with the data showing that the initial calcium transient in response to zymosan occurs in the absence of extracellular calcium (Fig. 9C).

The effects of chelating agents and thapsigargin on phagocytosis of non-opsonized zymosan were evaluated by determining the percentage of cells undergoing phagocytosis and the phagocytic index (Fig. 9F). BAPTA-AM pretreatment markedly reduced the number of cells undergoing phagocytosis and the phagocytic index (13 and 1% of control levels, respectively; controls were macrophages undergoing phagocytosis in CCB). In cells incubated in medium containing both BAPTA-AM and EGTA, to chelate intracellular and extracellular calcium, the percent of phagocytic cells and the phagocytic index were further decreased to 5 and 0.2%, respectively. In the presence of EGTA the number of phagocytic cells was reduced to 53% and the phagocytic index to 12% of control levels, indicating that abolishing the sustained phase impaired phagocytosis. However, limited phagocytosis of one or two particles was still observed in some cells after the initial calcium transient had returned to basal levels. Under conditions of extracellular calcium chelation, [Ca2+]i remained at basal levels in cells undergoing 1 event of phagocytosis (Fig. 9C, red line). In all cases of phagocytosis examined under these conditions, GFP-cPLA2{alpha} translocated to the phagosome (Fig. 9C, inset; the image was taken at the time indicated by the downward arrow). In cells pretreated with thapsigargin, the number of phagocytic cells and the phagocytic index were reduced, respectively, to 45 and 9% of control levels; however, as in the case of cells incubated in extracellular EGTA, when limited phagocytosis was detected in cells treated with thapsigargin, cPLA2{alpha} translocated to the forming phagosome.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Resident tissue macrophages are a first line of defense in innate immunity against microbial infection. Professional phagocytes engulf and kill microorganisms and present antigen for triggering adaptive immune responses (47-49). During phagocytosis macrophages secrete preformed granule constituents and newly synthesized products that play diverse roles in inflammation and tissue repair. Zymosan is a potent inducer of acute inflammation in animal models initiated in part by activation of macrophages and production of eicosanoids (50, 51). Resident mouse peritoneal macrophages have been used extensively as a model to study the regulation of arachidonic acid release and eicosanoid production induced by zymosan (13, 30, 33, 52-55). They contain a high content of esterified arachidonic acid, which is specifically released in response to zymosan, and converted to PGE2 and leukotriene C4 (12, 56). Eicosanoids mediate acute inflammatory responses such as increased vascular permeability and recruitment of leukocytes to the site of infection (57).

The production of eicosanoids by zymosan-treated macrophages is initiated by activation of cPLA2{alpha}, which must be recruited to intracellular membranes for release of arachidonic acid from phospholipid (13). In macrophages treated with ionomycin, cPLA2{alpha} translocates to Golgi, ER, and NM as observed in other cell types. However, during phagocytosis of zymosan, cPLA2{alpha} rapidly translocates to the forming phagosome and to ruffle-like regions of the plasma membrane. Macrophages are capable of ingesting numerous, large particles such as yeast, which requires more membrane than can be provided by plasma membrane for phagosome formation (32, 45, 58, 59). cPLA2{alpha} rapidly surrounded the zymosan particle and overlapped completely with the early endosome marker Rab5 and the plasma membrane cell surface macrophage marker, F4/80. Rab5 is recruited to the forming phagosome during phagocytosis of opsonized and non-opsonized particles and is required for fusion events leading to formation of the phagolysosome (60). Our results demonstrate that translocation of cPLA2{alpha} to the phagosome is an early event that occurs with similar kinetics as acquisition of early endosome markers and that precedes phagolysosome formation.

The recruitment of ER membrane to the forming phagosome during zymosan phagocytosis was shown using antibodies to COX2 and GRP78, resident ER proteins. It has previously been reported that ER proteins are observed on the 15-min phagocytic cup/phagosome in macrophages ingesting latex beads and microorganisms (31). We observed that GRP78 and COX2 localized to the region of the phagocytic cup 5 min after zymosan internalization and surrounded the particle by 15 min. Localization of cPLA2{alpha} to the forming phagosome appeared to precede fusion with the ER, and cPLA2{alpha} was present on extensive regions of the phagosome that were devoid of ER markers. The results indicate that the localization of cPLA2{alpha} to the phagosome is not because of targeting to ER. The localization of cPLA2{alpha} followed by COX2 to the phagosome correlated with the time course of PGE2 production suggesting that the phagosome membrane may serve as a site for cPLA2{alpha}-mediated release of arachidonic acid for prostanoid production through the COX2 pathway.

During zymosan phagocytosis, cPLA2{alpha} localizes to the forming phagosome and to ruffles but not to other regions of the plasma membrane suggesting that cPLA2{alpha} targets components formed at these sites. Membrane ruffles are involved in cell migration and their formation involves actin remodeling and activation of Rac1, which are regulated by phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, respectively (61, 62). Actin remodeling and phosphatidylinositol 3-kinases are also involved in pseudopod extension during phagocytosis (63). It has recently been shown that arachidonic acid can promote phagosome actin assembly in an in vitro assay (64). We have found that zymosan is internalized in macrophages treated with pyrrolidine, a cPLA2{alpha} inhibitor that dramatically reduces arachidonic acid release in response to zymosan, suggesting that cPLA2{alpha} action is not required for phagosome formation (14).

Calcium has been implicated as a regulator of phagocytosis, however, its role in mediating the sequence of events occurring during phagocytosis initiated by particle binding to different phagocytic receptors is not understood. In elicited mouse peritoneal macrophages it was found that both IgG-coated and un-coated latex beads induced a rapid, transient increase in [Ca2+]i in single cells that preceded phagocytosis. The calcium increase was required for phagocytosis although the regulation of the calcium increase and its role in phagocytosis differed between Fc receptor-mediated and "nonspecific" phagocytosis (37). These results and other studies suggest that the regulation of calcium mobilization and its role in phagocytosis differs depending on the receptors involved in mediating particle uptake. We previously reported that zymosan induced a transient increase in [Ca2+]i in macrophages and that chelating extracellular and intracellular calcium significantly decreased zymosan-induced arachidonic acid release (30). We have extended these observations by quantitating changes in [Ca2+]i over time during the course of zymosan uptake in single cells and determining whether calcium was required for regulating zymosan ingestion and translocation of cPLA2{alpha}. We found that zymosan induces a biphasic increase in [Ca2+]i, an initial high amplitude transient increase followed by a low amplitude sustained increase. The initial phase was due to release from intracellular stores because it was un-affected by chelating extracellular calcium but was blocked by thapsigargin. It occurred within 15-30 s of particle addition and preceded zymosan internalization. The sustained calcium increase was due to an influx from extracellular sources since it was blocked by chelating extracellular calcium. Phagocytosis of zymosan was prevented by chelating both intracellular and extracellular calcium although particles were observed bound to the cell surface. Binding of zymosan to the {beta}-glucan receptor dectin-1, which mediates uptake of zymosan by macrophages, has been reported to be calcium-independent (7). The results demonstrate that calcium is required for the internalization of zymosan.

Investigating the role of calcium in mediating cPLA2{alpha} translocation to phagosomes is complicated because calcium plays a role in zymosan internalization. However, limited phagocytosis could be detected under conditions of extracellular calcium chelation, which prevented the low amplitude sustained increase in calcium. In this case, whenever phagocytosis was detected, GFP-cPLA2{alpha} translocated to the phagosome. Since translocation to the phagosome occurred when [Ca2+]i was at basal levels in macrophages incubated in the presence of EGTA, it suggests that an increase in [Ca2+]i may not be the immediate effector of cPLA2{alpha} translocation to phagosomes. However, this does not preclude a functional role for basal levels of calcium that may be required for stable binding of cPLA2{alpha} to the membrane. This would be consistent with our previous work showing that basal levels of intracellular calcium and a functional C2 domain are required for cPLA2{alpha}-mediated arachidonic acid release in cells treated with agonists that do not mobilize calcium such as phorbol 12-myristate 13-acetate and okadaic acid (16, 21, 30, 33, 65). The mechanism involved in targeting cPLA2{alpha} to membrane without an increase in [Ca2+]i is not known but may involve production of membrane components that increase the affinity of cPLA2{alpha} for calcium. One possibility may involve formation of polyphosphoinositides. We have previously reported that phosphatidylinositol 4,5-bisphosphate activates cPLA2{alpha} and increases its affinity for calcium (66). Phosphatidylinositol 4,5-bisphosphate also promotes binding of cPLA2{alpha} to phospholipid vesicles and a role for the catalytic domain has been suggested (67, 68). It is therefore interesting to speculate that formation of polyphosphoinositides at the phagosome membrane and in ruffles may play a role in localization of cPLA2{alpha} at these sites.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL34303 and HL61378. 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. Back

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Fig. 1 and videos 1-3. Back

|| To whom correspondence should be addressed: Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1214; Fax: 303-270-2155; E-mail: lesliec{at}njc.org.

1 The abbreviations used are: COX1 and COX2, cyclooxygenase 1 and 2; NM, nuclear membrane; CCB, complete calcium buffer; cPLA2{alpha}, cytosolic phospholipase A2 {alpha}; BAPTA-AM, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester); [Ca2+]i, intracellular calcium concentration; ER, endoplasmic reticulum; PGE2, prostaglandin E2; GFP, green fluorescent protein; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; AdGFP, adenovirus green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank William Townend for assistance with the time-lapse microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gordon, S. (2002) Cell 111, 927-930[CrossRef][Medline] [Order article via Infotrieve]
  2. Underhill, D. M., and Ozinsky, A. (2002) Annu. Rev. Immunol. 20, 825-852[CrossRef][Medline] [Order article via Infotrieve]
  3. Di Carlo, F. J., and Fiore, J. V. (1957) Science 127, 756-757
  4. Xia, Y., Vetvicka, V., Yan, J., Hanikyrova, M., Mayadas, T., and Ross, G. D. (1999) J. Immunol. 162, 2281-2290[Abstract/Free Full Text]
  5. Brown, G. D., Taylor, P. R., Reid, D. M., Willment, J. A., Williams, D. L., Jr., Marinez-Pomares, L., Wong, S. Y., and Gordon, S. (2002) J. Exp. Med. 196, 407-412[Abstract/Free Full Text]
  6. Ezekowitz, R. A., Sastry, K., Bailly, P., and Warner, A. (1990) J. Exp. Med. 172, 1785-1794[Abstract/Free Full Text]
  7. Brown, G. D., and Gordon, S. (2001) Nature 413, 36-37[Medline] [Order article via Infotrieve]
  8. Gantner, B. N., Simmons, R. M., Canavera, S. J., Akira, S., and Underhill, D. M. (2003) J. Exp. Med. 197, 1107-1117[Abstract/Free Full Text]
  9. Brown, G. D., Herre, J., Williams, D. L., Jr., Willment, J. A., Marshall, A. S., and Gordon, S. (2003) J. Exp. Med. 197, 1119-1124[Abstract/Free Full Text]
  10. Ozinsky, A., Underhill, D. M., Fontenot, J. D., Hajjar, A. M., Smith, K. D., Wilson, C. B., Schroeder, L., and Aderem, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13766-13771[Abstract/Free Full Text]
  11. Humes, J. L., Bonney, R. J., Pelus, L., Dahlgren, M. E., Sadowski, S. J., Kuehl, F. A., Jr., and Davies, P. (1977) Nature 269, 149-151[CrossRef][Medline] [Order article via Infotrieve]
  12. Rouzer, C. A., Scott, W. A., Cohn, Z. A., Blackburn, P., and Manning, J. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4928-4932[Abstract/Free Full Text]
  13. Gijón, M. A., Spencer, D. M., Siddiqi, A. R., Bonventre, J. V., and Leslie, C. C. (2000) J. Biol. Chem. 275, 20146-20156[Abstract/Free Full Text]
  14. Ghomashchi, F., Stewart, A., Hefner, Y., Ramanadham, S., Turk, J., Leslie, C. C., and Gelb, M. H. (2001) Biochim. Biophys. Acta 1513, 160-166[Medline] [Order article via Infotrieve]
  15. Leslie, C. C. (1997) J. Biol. Chem. 272, 16709-16712[Free Full Text]
  16. Gijón, M. A., Spencer, D. M., and Leslie, C. C. (2000) Adv. Enzyme Regul. 40, 255-268[CrossRef][Medline] [Order article via Infotrieve]
  17. Lin, L.-L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278[CrossRef][Medline] [Order article via Infotrieve]
  18. Hefner, Y., Borsch-Haubold, A. G., Murakami, M., Wilde, J. I., Pasquet, S., Schieltz, D., Ghomashchi, F., Yates, J. R., 3rd, Armstrong, C. G., Paterson, A., Cohen, P., Fukunaga, R., Hunter, T., Kudo, I., Watson, S. P., and Gelb, M. H. (2000) J. Biol. Chem. 275, 37542-37551[Abstract/Free Full Text]
  19. Muthalif, M. M., Hefner, Y., Canaan, S., Harper, J., Zhou, H., Parmentier, J.-H., Aebersold, R., Gelb, M. H., and Malik, K. U. (2001) J. Biol. Chem. 276, 39653-39660[Abstract/Free Full Text]
  20. de Carvalho, M. G. S., McCormack, A. L., Olson, E., Ghomashchi, F., Gelb, M. H., Yates, J. R., III, and Leslie, C. C. (1996) J. Biol. Chem. 271, 6987-6997[Abstract/Free Full Text]
  21. Gijón, M. A., Spencer, D. M., Kaiser, A. L., and Leslie, C. C. (1999) J. Cell Biol. 145, 1219-1232[Abstract/Free Full Text]
  22. Schievella, A. R., Regier, M. K., Smith, W. L., and Lin, L.-L. (1995) J. Biol. Chem. 270, 30749-30754[Abstract/Free Full Text]
  23. Glover, S., de Carvalho, M. S., Bayburt, T., Jonas, M., Chi, E., Leslie, C. C., and Gelb, M. H. (1995) J. Biol. Chem. 270, 15359-15367[Abstract/Free Full Text]
  24. Evans, J. H., Spencer, D. M., Zweifach, A., and Leslie, C. C. (2001) J. Biol. Chem. 276, 30150-30160[Abstract/Free Full Text]
  25. Peters-Golden, M., and Brock, T. G. (2001) FEBS Lett. 487, 323-326[CrossRef][Medline] [Order article via Infotrieve]
  26. Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 145-182[CrossRef][Medline] [Order article via Infotrieve]
  27. Spencer, A. G., Woods, J. W., Arakawa, T., Singer, I. I., and Smith, W. L. (1998) J. Biol. Chem. 273, 9886-9893[Abstract/Free Full Text]
  28. Sierra-Honigmann, M. R., Bradley, J. R., and Pober, J. S. (1996) Lab. Investig. 74, 684-695[Medline] [Order article via Infotrieve]
  29. Shmelzer, Z., Haddad, N., Admon, E., Pessach, I., Leto, T. L., Eitan-Hazan, Z., Hershfinkel, M., and Levy, R. (2003) J. Cell Biol. 162, 683-692[Abstract/Free Full Text]
  30. Qiu, Z.-H., Gijón, M. A., de Carvalho, M. S., Spencer, D. M., and Leslie, C. C. (1998) J. Biol. Chem. 273, 8203-8211[Abstract/Free Full Text]
  31. Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., Steele-Mortimer, O., Paiement, J., Bergeron, J. J., and Desjardins, M. (2002) Cell 110, 119-131[CrossRef][Medline] [Order article via Infotrieve]
  32. Desjardins, M. (2003) Nat. Rev. Immunol. 3, 280-291[CrossRef][Medline] [Order article via Infotrieve]
  33. Qiu, Z.-H., de Carvalho, M. S., and Leslie, C. C. (1993) J. Biol. Chem. 268, 24506-24513[Abstract/Free Full Text]
  34. de Carvalho, M. S., McCormack, F. X., and Leslie, C. C. (1993) Arch. Biochem. Biophys. 306, 534-540[CrossRef][Medline] [Order article via Infotrieve]
  35. Stewart, A., Ghosh, M., Spencer, D. M., and Leslie, C. C. (2002) J. Biol. Chem. 277, 29526-29536[Abstract/Free Full Text]
  36. Evans, J. H., Fergus, D. J., and Leslie, C. C. (2002) BMC Biochemistry http://www.biomedcentral.com/1471-2091/3/30
  37. Hishikawa, T., Cheung, J. Y., Yelamarty, R. V., and Knutson, D. W. (1991) J. Cell Biol. 115, 59-66[Abstract/Free Full Text]
  38. Larsen, E. C., DiGennaro, J. A., Saito, N., Mehta, S., Loegering, D. J., Mazurkiewicz, J. E., and Lennartz, M. R. (2000) J. Immunol. 165, 2809-2817[Abstract/Free Full Text]
  39. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract/Free Full Text]
  40. Balla, T., and Varnai, P. (2002) Science's STKE http://www.stke.org/cgi/content/full/OC_sigtrans;2002/PL3
  41. Gordon, S., Crocker, P. R., Morris, L., Lee, S. H., Perry, V. H., and Hume, D. A. (1986) Ciba Found. Symp. 118, 54-67[Medline] [Order article via Infotrieve]
  42. Taylor, P. R., Brown, G. D., Geldhof, A. B., Martinez-Pomares, L., and Gordon, S. (2003) Eur. J. Immunol. 33, 2090-2097[CrossRef][Medline] [Order article via Infotrieve]
  43. Desjardins, M., Huber, L. A., Parton, R. G., and Griffiths, G. (1994) J. Cell Biol. 124, 677-688[Abstract/Free Full Text]
  44. Desjardins, M., Celis, J. E., van Meer, G., Dieplinger, H., Jahraus, A., Griffiths, G., and Huber, L. A. (1994) J. Biol. Chem. 269, 32194-32200[Abstract/Free Full Text]
  45. Vieira, O. V., Botelho, R. J., and Grinstein, S. (2002) Biochem. J. 366, 689-704[CrossRef][Medline] [Order article via Infotrieve]
  46. Alvarez-Dominguez, C., Barbieri, A. M., Beron, W., Wandinger-Ness, A., and Stahl, P. D. (1996) J. Biol. Chem. 271, 13834-13843[Abstract/Free Full Text]
  47. Hume, D. A., Ross, I. L., Himes, S. R., Sasmono, R. T., Wells, C. A., and Ravasi, T. (2002) J. Leukocyte Biol. 72, 621-627[Abstract/Free Full Text]
  48. Gordon, S. (2003) Nat. Rev. Immunol. 3, 23-35[CrossRef][Medline] [Order article via Infotrieve]
  49. Mosser, D. M. (2003) J. Leukocyte Biol. 73, 209-212[Free Full Text]
  50. Doherty, N. S., Poubelle, P., Borgeat, P., Beaver, T. H., Westrich, G. L., and Schrader, N. L. (1985) Prostaglandins 30, 769-775[CrossRef][Medline] [Order article via Infotrieve]
  51. Lundy, S. R., Dowling, R. L., Stevens, T. M., Kerr, J. S., Mackin, W. M., and Gans, K. R. (1990) J. Immunol. 144, 2671-2677[Abstract]
  52. Emilsson, A., and Sundler, R. (1986) Biochim. Biophys. Acta 876, 533-542[Medline] [Order article via Infotrieve]
  53. Bonney, R. J., Wightman, P. D., Davies, P., Sadowski, S. J., Kuehl, F. A., Jr., and Humes, J. L. (1978) Biochem. J. 176, 433-442[Medline] [Order article via Infotrieve]
  54. Hiller, G., and Sundler, R. (1999) Cell. Signal. 11, 863-869[CrossRef][Medline] [Order article via Infotrieve]
  55. Aderem, A. A., Scott, W. A., and Cohn, Z. A. (1986) J. Exp. Med. 163, 139-154[Abstract/Free Full Text]
  56. Scott, W. A., Zrike, J. M., Hamill, A. L., Kempe, J., and Cohn, Z. A. (1980) J. Exp. Med. 152, 324-335[Abstract/Free Full Text]
  57. Funk, C. D. (2001) Science 294, 1871-1875[Abstract/Free Full Text]
  58. Aderem, A., and Underhill, D. M. (1999) Annu. Rev. Immunol. 17, 593-623[CrossRef][Medline] [Order article via Infotrieve]
  59. Aderem, A. (2002) Cell 110, 5-8[CrossRef][Medline] [Order article via Infotrieve]
  60. Duclos, S., Diez, R., Garin, J., Papadopoulou, B., Descoteaux, A., Stenmark, H., and Desjardins, M. (2000) J. Cell Sci. 113, 3531-3541[Abstract]
  61. Lauffenburger, D. A., and Horwitz, A. F. (1996) Cell 84, 359-369[CrossRef][Medline] [Order article via Infotrieve]
  62. Small, J. V., Stradal, T., Vignal, E., and Rottner, K. (2002) Trends Cell Biol. 12, 112-120[CrossRef][Medline] [Order article via Infotrieve]
  63. Greenberg, S. (1999) J. Leukocyte Biol. 66, 712-717[Abstract]
  64. Anes, E., Kuhnel, M. P., Bos, E., Moniz-Pereira, J., Habermann, A., and Griffiths, G. (2003) Nat. Cell Biol. 5, 793-802[CrossRef][Medline] [Order article via Infotrieve]
  65. Gijón, M. A., and Leslie, C. C. (1999) J. Leukocyte Biol. 65, 330-336[Abstract]
  66. Leslie, C. C., and Channon, J. Y. (1990) Biochim. Biophys. Acta 1045, 261-270[Medline] [Order article via Infotrieve]
  67. Mosior, M., Six, D. A., and Dennis, E. A. (1998) J. Biol. Chem. 273, 2184-2191[Abstract/Free Full Text]
  68. Six, D. A., and Dennis, E. A. (2003) J. Biol. Chem. 278, 23842-23850[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
B. Balestrieri, A. Maekawa, W. Xing, M. H. Gelb, H. R. Katz, and J. P. Arm
Group V Secretory Phospholipase A2 Modulates Phagosome Maturation and Regulates the Innate Immune Response against Candida albicans
J. Immunol., April 15, 2009; 182(8): 4891 - 4898.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Shmelzer, M. Karter, M. Eisenstein, T. L. Leto, N. Hadad, D. Ben-Menahem, D. Gitler, S. Banani, B. Wolach, M. Rotem, et al.
Cytosolic Phospholipase A2{alpha} Is Targeted to the p47phox-PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain
J. Biol. Chem., November 14, 2008; 283(46): 31898 - 31908.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
Y. Tian, C. Pate, A. Andreolotti, L. Wang, E. Tuomanen, K. Boyd, E. Claro, and S. Jackowski
Cytokine secretion requires phosphatidylcholine synthesis
J. Cell Biol., October 21, 2008; 181(6): 945 - 957.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
D. E. Tucker, M. A. Gijon, D. M. Spencer, Z.-H. Qiu, M. H. Gelb, and C. C. Leslie
Regulation of cytosolic phospholipase A2{alpha} by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages
J. Leukoc. Biol., September 1, 2008; 84(3): 798 - 806.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Noor, H. Goldfine, D. E. Tucker, S. Suram, L. L. Lenz, S. Akira, S. Uematsu, M. Girotti, J. V. Bonventre, K. Breuel, et al.
Activation of Cytosolic Phospholipase A2{alpha} in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2
J. Biol. Chem., February 22, 2008; 283(8): 4744 - 4755.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Yamashita, T. Shinohara, S. Tsuji, Q. N. Myrvik, A. Nishiyama, R. A. Henriksen, and Y. Shibata
Catalytically Inactive Cyclooxygenase 2 and Absence of Prostaglandin E2 Biosynthesis in Murine Peritoneal Macrophages following In Vivo Phagocytosis of Heat-Killed Mycobacterium bovis Bacillus Calmette-Guerin
J. Immunol., November 15, 2007; 179(10): 7072 - 7078.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Ghosh, R. Loper, F. Ghomashchi, D. E. Tucker, J. V. Bonventre, M. H. Gelb, and C. C. Leslie
Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2{zeta} in Mouse Lung Fibroblasts
J. Biol. Chem., April 20, 2007; 282(16): 11676 - 11686.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. Kolko, J. Wang, C. Zhan, K. A. Poulsen, J. U. Prause, M. H. Nissen, S. Heegaard, and N. G. Bazan
Identification of Intracellular Phospholipases A2 in the Human Eye: Involvement in Phagocytosis of Photoreceptor Outer Segments
Invest. Ophthalmol. Vis. Sci., March 1, 2007; 48(3): 1401 - 1409.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. S. Goodridge, R. M. Simmons, and D. M. Underhill
Dectin-1 Stimulation by Candida albicans Yeast or Zymosan Triggers NFAT Activation in Macrophages and Dendritic Cells
J. Immunol., March 1, 2007; 178(5): 3107 - 3115.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
T. Ueyama, T. Tatsuno, T. Kawasaki, S. Tsujibe, Y. Shirai, H. Sumimoto, T. L. Leto, and N. Saito
A Regulated Adaptor Function of p40phox: Distinct p67phox Membrane Targeting by p40phox and by p47phox
Mol. Biol. Cell, February 1, 2007; 18(2): 441 - 454.
[Abstract] [Full Text] [PDF]

Home page
 Eukaryot CellHome page
M. Liu, P. Du, G. Heinrich, G. M. Cox, and A. Gelli
Cch1 Mediates Calcium Entry in Cryptococcus neoformans and Is Essential in Low-Calcium Environments
Eukaryot. Cell, October 1, 2006; 5(10): 1788 - 1796.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
I. Pessach, Z. Shmelzer, T. L. Leto, M. C. Dinauer, and R. Levy
The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation
J. Leukoc. Biol., September 1, 2006; 80(3): 630 - 639.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Balestrieri, V. W. Hsu, H. Gilbert, C. C. Leslie, W. K. Han, J. V. Bonventre, and J. P. Arm
Group V Secretory Phospholipase A2 Translocates to the Phagosome after Zymosan Stimulation of Mouse Peritoneal Macrophages and Regulates Phagocytosis
J. Biol. Chem., March 10, 2006; 281(10): 6691 - 6698.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Suram, G. D. Brown, M. Ghosh, S. Gordon, R. Loper, P. R. Taylor, S. Akira, S. Uematsu, D. L. Williams, and C. C. Leslie
Regulation of Cytosolic Phospholipase A2 Activation and Cyclooxygenase 2 Expression in Macrophages by the beta-Glucan Receptor
J. Biol. Chem., March 3, 2006; 281(9): 5506 - 5514.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
O. H. Vandal, M. H. Gelb, S. Ehrt, and C. F. Nathan
Cytosolic Phospholipase A2 Enzymes Are Not Required by Mouse Bone Marrow-Derived Macrophages for the Control of Mycobacterium tuberculosis In Vitro
Infect. Immun., March 1, 2006; 74(3): 1751 - 1756.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
I. Beck-Speier, N. Dayal, E. Karg, K. L. Maier, G. Schumann, M. Semmler, and S. M. Koelsch
Oxymetazoline Inhibits Proinflammatory Reactions: Effect on Arachidonic Acid-Derived Metabolites
J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 843 - 851.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Interv.Home page
N. F. Lamour and C. E. Chalfant
Ceramide-1-phosphate: The "missing" link in eicosanoid biosynthesis and inflammation
Mol. Interv., December 1, 2005; 5(6): 358 - 367.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
H. Liu, A. M. Mamoon, and J. M. Farley, Sr.
Prostanoids Secreted by Alveolar Macrophages Enhance Ionic Currents in Swine Tracheal Submucosal Gland Cells
J. Pharmacol. Exp. Ther., November 1, 2005; 315(2): 729 - 739.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H.-J. van Manen, Y. M. Kraan, D. Roos, and C. Otto
Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes
PNAS, July 19, 2005; 102(29): 10159 - 10164.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
N. Touret, P. Paroutis, and S. Grinstein
The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma
J. Leukoc. Biol., June 1, 2005; 77(6): 878 - 885.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Shohdy, J. A. Efe, S. D. Emr, and H. A. Shuman
Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking
PNAS, March 29, 2005; 102(13): 4866 - 4871.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. B. Rubin, G. P. Downey, A. Koh, N. Degousee, F. Ghomashchi, L. Nallan, E. Stefanski, D. W. Harkin, C. Sun, B. P. Smart, et al.
Cytosolic Phospholipase A2-{alpha} Is Necessary for Platelet-activating Factor Biosynthesis, Efficient Neutrophil-mediated Bacterial Killing, and the Innate Immune Response to Pulmonary Infection: cPLA2-{alpha} DOES NOT REGULATE NEUTROPHIL NADPH OXIDASE ACTIVITY
J. Biol. Chem., March 4, 2005; 280(9): 7519 - 7529.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/18/19113    most recent
M313867200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Girotti, M.
Right arrow Articles by Leslie, C. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Girotti, M.
Right arrow Articles by Leslie, C. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement