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Lipoxin A4 activates ALX/FPR2 Receptor to Regulate Conjunctival Goblet Cell Secretion

Conjunctival goblet cells play a major role in maintaining the mucous layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis.. Resolution of inflammation is mediated by pro-resolution agonists such as lipoxin A4 (LXA4) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca2+] ([Ca2+]i) and identify signaling pathways activated by LXA4. ALX/FPR was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA4 significantly increased mucin secretion, [Ca2+]i, and ERK 1/2 activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA4 increased [Ca2+]i to the same extent as LXA4. Sequential addition of either LXA4 or resolvin D1 followed by the second compound decreased [Ca2+]i of the second compound compared to its initial response. LXA4 activated phospholipase C, -D, and A2 and downstream molecules protein kinase C, ERK 1/2, and Ca2+/calmodulin dependent kinase to increase mucin secretion and [Ca2+]i. We conclude that conjunctival goblet cells respond to LXA4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases.


INTRODUCTION
The cornea and conjunctiva comprise the ocular surface of the eye. Within the conjunctiva, goblet cells are interspersed throughout the stratified squamous cells. Goblet cells are responsible the synthesis and secretion of mucins into the tears and onto the ocular surface. Mucins, along with other components of the tear film, protect the cornea and conjunctiva from the external environment preventing dessication as well as adherence of bacteria and allergens. Recent studies have demonstrated that goblet cells play an active role in the innate immune response of the conjunctiva and are directly affected by cytokines produced during inflammation [1][2][3] . In the context of the ocular surface, the types of inflammation observed include seasonal allergic conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, giant papillary conjunctivitis, chemical and thermal burns and dry eye syndrome 4 . Uncontrolled inflammation is a hallmark of these diseases causing redness, itching, and discomfort.
Resolution of inflammation is an active process and occurs with the switch from the generation of pro-inflammatory mediators such as leukotrienes and prostaglandins to the generation of pro-resolution mediators such as the resolvins (Rv), lipoxins (LX), protectins, and maresins 5 . These compounds are known, collectively, as specialized proresolving lipid mediators (SPM) 5 . Lipoxins are biosynthesized from arachidonic acid during the resolution of inflammation. Two main types of lipoxins are produced in vivo: LXA 4 and LXB 4 . In the presence of aspirin, aspirin-triggered epimers of LXA 4 (ATL) and LXB 4 are formed 6 . In the conjunctiva, pretreatment of goblet cells with the pro-resolution compounds RvD1 and aspirin-triggered RvD1 (AT-RvD1) significantly decreased histamine-stimulated mucin secretion 7 . In addition, RvD1 and RvE1 were both effective in inhibiting LTD 4 -stimulation of glycoconjugate secretion 8 . In addition to their well-established role in resolution of acute and chronic inflammation, it is likely that SPMs also have a role in the normal, physiological state. The expression of the receptor for LXA 4 , ALX, is endogenously expressed in numerous tissues 9,10 . In the eye, LXA 4 and the enzymes responsible for its synthesis are expressed in a healthy, non-inflamed mouse cornea 11,12 .
The role of LXA 4 under normal, physiological conditions in the conjunctiva is not known. Therefore, we explored the role of LXA 4 has an effect on conjunctival goblet cell glycoconjugate secretion and [Ca 2+ ] i and the effects of several stable analogs of LXA 4 on [Ca 2+ ] i in the absence of pro-inflammatory mediators. We demonstrate that LXA 4 responses are mediated via its receptor ALX/FPR2 to activate phospholipases -C, -D, and -A2 which in turn stimulates the downstream signaling molecules protein kinase C (PKC), extracellular regulated kinase (ERK 1/2), and calcium/calmodulin-dependent protein kinase (Ca 2+ / CaMK) to increase [Ca 2+ ] i and glycoconjugate secretion.

ALX/FPR2 receptor is present in cultured rat conjunctival goblet cells
To determine if ALX/FPR2 receptor is present in goblet cells, RT-PCR was performed using cDNA isolated from rat conjunctival goblet cells. A single band at the expected number of base pairs was observed ( Figure 1A). ALX/FPR2 was also detected in cultured goblet cells by western blot analysis using an antibody directed against ALX/FPR2 ( Figure 1B). A band at the expected molecular weight of 38 kDa was seen as well as a band at approximately 75 kDa which is consistent with ALX dimers 13 . To determine the location of ALX/FPR2 within the conjunctiva, immunofluorescence microscopy experiments were performed. In rat conjunctiva, ALX/FPR2 (shown in red, Figure 1C) was present in goblet cells and stratified squamous cells. The lectin UEA-1 (shown in green, Figure 1C), which binds to the secretory products of goblet cells, was used to identify the goblet cells. In cultured rat goblet cells, ALX/FPR2 (shown in red) was present throughout the cytosol ( Figure 1D). UEA-1, shown in green was used to confirm that the cultured cells were indeed goblet cells ( Figure 1D). There was significant overlap in the localization of ALX/FPR2 and UEA-1. Thus the presence of ALX/FPR2 was demonstrated in rat conjunctiva and goblet cells by RT-PCR, western blot analysis, and immunofluorescence microscopy.
To demonstrate that the rise in [Ca 2+ ] i stimulated by LXA 4 leads to glycoconjugate secretion goblet cells were incubated with the calcium chelator BAPTA/AM and glycoconjugate secretion measured. To determine the concentration of BAPTA/AM necessary to chelate [Ca 2+ ] i , cells were preincubated with BAPTA/AM from 10 −6 to 10 −4 M for 30 min and fura2 for 1h. The increase in [Ca 2+ ] i was then measured in response to LXA 4 (10 −9 M). LXA 4 significantly increased Ca 2+ i to 112 ± 15 nM (p=0.0003). Histamine (10 −5 M) was also used as a positive control as histamine has also been shown to stimulate an increase in [Ca 2+ ] i and secretion in these cells 14,15 . In these experiments, histamine significantly increased [Ca 2+ ] i to 265 ± 39 nM (p=0.0005). The increases in peak [Ca 2+ ] i in response to both LXA 4 and histamine response were significantly inhibited (p=0.05 and p=0.002, respectively) at 10 −5 M BAPTA/AM ( Figure 3A).
The result of these experiments indicates that similar to cholinergic agonists, histamine and RvD1 and AT-RvD1, LXA 4 alone stimulates secretion, an increase in [Ca 2+ ] i , and ERK 1/2 activation in cultured conjunctival goblet cells through the ALX receptor.

LXA 4 Analogs on [Ca 2+ ] i and Their Effects on LXA 4 -stimualted Increase in [Ca 2+ ] i
Because LXA 4 undergoes rapid inactivation, several, more stable, LXA 4 analogs have been synthesized. We tested the effects of several analogs on their ability to increase [Ca 2+ ] i in goblet cells and their effects on LXA 4 -stimulated increase in [Ca 2+ ] i . 15-epi-16-phenoxy-LXA 4 and 15-epi-16-parafluorophenoxy-LXA 4 , which have each been shown to prevent neutrophil recruitment 16 and inflammation 17 , were compared to LXA 4 , ATL and LTB 4 . All compounds (10 −9 M) increased [Ca 2+ ] i to a similar extent as LXA 4 and with a similar time frame to reach peak [Ca 2+ ] i (Table I). If the analogs were given 2 min prior to LXA 4 , the LXA 4 -stimulated increase in [Ca 2+ ] i was significantly decreased when compared to the LXA 4 response when added first (Table I). These results indicate that LXA 4 analogs could bind to the same or overlapping binding sites on ALX causing a desensitization of the receptor to a subsequent addition of LXA 4 .

Interaction of LXA 4 , RvD1, and Annexin A1 Binding to ALX/FPR2
The ALX/FPR2 receptor has multiple agonists which can bind to it. These agonists include the resolvin RvD1, the protein annexin A1 (AnxA1), as well as LXA 4 18 . We explored the interaction between the ligands of ALX/FPR2 in goblet cells using the following experimental paradigm: addition of first agonist followed 2 minutes later by addition of second agonist. Increase in [Ca 2+ ] i was measured after addition of each agonist. In rat goblet cells, an initial addition of RvD1 (10 −8 M) resulted in an significant increase in peak [Ca 2+ ] i of 209.6 ± 64.0 nM (p=0.01) ( Figure 6A and B). There was no additional increase in [Ca 2+ ] i after a second addition of RvD1. The same result was obtained if LXA 4 is added first followed by a subsequent addition of RvD1 ( Figure 6A and B). The second RvD1 response after initial addition of either RvD1 or LXA 4 was significantly decreased (p= 0.005and p=0.02, respectively) from the result obtained when RvD1 is added first.
If LXA 4 (10 −9 M) is added first, the initial response resulted in a significant change (p=0.02) in peak [Ca 2+ ] i of 255.2 ± 107.9 nM. A second addition of LXA 4 did not result in any increase in [Ca 2+ ] i ( Figure 6A and B). This is also true if RvD1 is given before LXA 4 . Therefore the second LXA 4 response after initial addition of either LXA 4 or RvD1 was significantly decreased (p= 0.01 and p= 0.01 respectively) from the result obtained when LXA 4 is added first.
These results indicate that, in rat goblet cells, RvD1 and LXA 4 could bind to the same or to overlapping sites causing a desensitization of the receptor to a subsequent addition of either of these two SPMs.
When AnxA1 (10 −8 M) was added first, the change in peak [Ca 2+ ] i was significantly increased over baseline by 309.2 ± 68.2 nM (p=0.001) ( Figure 6C and D). When AnxA1 was added again, 2 min after the first addition, the change in peak [Ca 2+ ] i was 47.8 ± 19.0 nM, which was significantly decreased (p=0.004) from the response when AnxA1 added first. Similar results were obtained when AnxA1 was added after RvD1 (10 −8 M) or LXA 4 (10 −9 M) as the change in peak [Ca 2+ ] i significantly decreased (p= 0.02 and p=0.05, respectively) to 89.7 ± 16.8 and 80.7 ± 70.8 nM, respectively ( Figures 6E and F). These decreases were significantly different from the response obtained when AnxA1 was added first.
These data indicate that AnxA1 alters the interactions of LXA 4 and RvD1 to the ALX/FPR2 receptor.

Signaling Pathways Activated by LXA 4 to Increase [Ca 2+ ] i and Glycoconjugate Secretion
ALX/FPR2 is known to bind multiple ligands implying that this receptor is coupled to a diverse array of signaling pathways. In addition to generation of IP 3 , activation of PLC also leads to activation of protein kinase C (PKC) through the release of diacylglycerol. When goblet cells were preincubated with PKC inhibitor Ro317649 (10 −7 M), LXA 4 -stimulated increase in [Ca 2+ ] i was significantly blocked by 54.4 ± 12.1% (p=0.02). Glycoconjugate secretion was completely blocked by Ro317649 ( Figure 7A and B).
As Cch has been shown to activate PLD in other tissues [21][22][23][24] , we used Cch stimulation of glycoconjugate secretion as a control for the effectiveness of PLD inhibitor. 1-But, but not tbut, completely blocked Cch-stimulated glycoconjugate secretion (data not shown).
To determine if LXA 4 increases cAMP levels, goblet cells were incubated with the PKA inhibitor H89 (10 −5 M). H89 did not alter either LXA 4 -stimulated increase in [Ca 2+ ] i or glycoconjugate secretion ( Figure 7E and F)

Discussion
In the present study, we showed that in goblet cells activation of ALX/FPR2 by LXA 4 induces a number of signaling components involved in cellular pathways leading to an increase in [Ca 2+ ] i and mucin secretion. These signaling components include the phospholipases -C, -D, and -A2 and a number of downstream molecules, ERK 1/2, PKC, and Ca 2+ /CaMK but not PKA ( Figure 8A). ALX/FPR2 is known to bind a wide variety of ligand types including peptides, lipids, and small molecules including LXA 4 18, 28 . This receptor has been shown to have interactions with multiple G proteins after binding the peptide N-formyl-methionyl-leucyl-phenylalanine 29 . This allows for the coupling of a variety of signaling molecules which include phospholipase A2 and D, ERK 1/2, JNK, and p38 29 . One pathway activated in conjunctival goblet cells is the PLC pathway. Activation of this pathway induced release of Ca 2+ i by activating the IP 3 receptor and PKC. The increase in [Ca 2+ ] i subsequently activated Ca 2+ /CaMK. As PLD and PLA2 also increased the [Ca 2+ ] i , these enzymes could have also activated Ca 2+ /CaMK. In support of this suggestion is our finding that inhibition of Ca 2+ /CaMK completely blocked LXA 4 -stimulated increase in [Ca 2+ ] i and secretion. In contrast to Ca 2+ /CaMK, blockage of the IP 3 receptor, although it significantly inhibited secretion, is not as effective a blockade of secretion as is the Ca 2+ / CaMK inhibitor. This finding is consistent with only PLC, not PLD or PLA2, activating the IP 3 receptor.
Inhibition of PKC or ERK1/2 caused a different effect than inhibition of Ca 2+ /CaMK. Inhibition of PKC or ERK1/2 did not completely suppress LXA 4 -induced increase in [Ca 2+ ] i, but completely impaired LXA 4 stimulation of secretion. These results suggest that PKC and ERK1/2, when activated, use both Ca 2+ -dependent and Ca 2+ independent pathways to induce secretion. PKC consists of a family of multiple isoforms that are divided into three types: 1) DAG and Ca 2+ -dependent isoforms 2) DAG dependent but Ca 2+independent isoforms, and 3) DAG and Ca 2+ -independent isoforms 34 . ERK 1/2 can also be activated in a Ca 2+ independent manner [35][36][37] . Our findings suggest that LXA 4 activates multiple isoforms of PKC with at least one being a Ca 2+ -dependent isoform and one Ca 2+independent isoform. These data also indicate that ERK 1/2 activation by LXA 4 occurs via both Ca 2+ -dependent and Ca 2+ -independent mechanisms. LXA 4 in our studies does not activate PKA to increase [Ca 2+ ] i nor to stimulate secretion. In conjunctival goblet cells VIP uses cAMP and PKA to stimulate mucin secretion and does so in a Ca 2+ -dependent manner 38 . This is consistent with activation of Ca 2+ -dependent isoforms of adenylyl cyclase. In spite of the fact the LXA 4 in the present study increases [Ca 2+ ] i, this agonist appears not to be coupled to Gαs and adenylyl cyclase.
Interestingly, LXA 4 has an effect on goblet cells alone in the absence of any proinflammatory molecules as these experiments in this study were performed in vitro. Similar results were obtained with RvD1 and AT-RvD1 7 . These resolvins increased [Ca 2+ ] i , glycoconjugate secretion, and short term activation of ERK 1/2 (less than 30 min) but blocked histamine-stimulated actions 7 over longer times. We hypothesize that this activation by SPMs including LXA 4 is vital to maintaining the normal homeostasis of the ocular surface. This would imply that regulation of SPM biosynthesis within goblet cells must be tightly controlled.
The role of LXA 4 in the goblet cells is very complicated. Under normal, physiological conditions, endogenous LXA 4 likely stimulates mucin secretion to maintain the barrier functions and antimicrobial activities necessary to protect the cornea. Under inflammatory conditions in which there is excess mucin secretion LXA 4 likely plays an anti-inflammatory function to reduce mucin secretion. Similarly, LXA 4 is endogenously expressed in the cornea without inflammation present and the amount of LXA 4 was increased after corneal wounding 12 . Exogenous addition of LXA 4 promoted wound healing 12 and increased goblet cell number and mucin area in a desiccating stress model of dry eye 39 It is not known if the conjunctiva produces LXA 4 but it is possible that LXA 4 produced by the cornea could diffuse via tears to activate goblet cells.
Activation of ALX/FPR2 in goblet cells with either LXA 4 or RvD1 blocked a subsequent addition of either of these SPMs. This could be due to the fact that the receptor has desensitized. Maderna et al has shown that ALX/FPR2 is internalized after treatment with LXA 4 though the maximum internalization did not occur until 30 min after the addition 41 . It is also possible that LXA 4 and RvD1 bind to the same or overlapping binding sites in the receptor. LXA 4 was shown to bind to the extracellular loop III and the related transmembrane area 42 . The location to which RvD1 binds to ALX/FPR2 has not yet been determined.
In goblet cells, addition of AnxA1 or LXA 4 blocks the increase in [Ca 2+ ] i stimulated by a second addition of either agonist. This would be consistent with AnxA1 and LXA 4 binding to the same or overlapping sites. Interestingly, while an initial addition of RvD1 blocked the increase in [Ca 2+ ] i to a subsequent addition of AnxA1, the peak [Ca 2+ ] i stimulated by RvD1 after addition of AnxA1 was significantly increased compared to the response obtained when RvD1 was added first. Binding of ligands to ALX is necessarily complicated as the receptor can bind proteins, peptides and lipids 28 and can form homo-and heterodimers 13 . LXA 4 and ATL are rapidly synthesized in response to stimuli and are also rapidly degraded 16 . Stable analogs of these compounds would be advantageous for long term treatments. We tested two such analogs and compared them to LXA 4 , ATL, and LTB 4 . All analogs behaved similarly to LXA 4 in that intracellular [Ca 2+ ] was mobilized to similar extents. In addition, as all compounds blocked a second addition of LXA 4 , it is likely that all bind to ALX, similar to LXA 4 .
Several SPMs including LXA 4 , RvD1, and RvE1 stimulate conjunctival goblet cell secretion. These three SPMs are biosynthesized by three different pathways. LXA 4 is derived from arachidonic acid, RvD1 from docsahexaenoic acid, and RvE1 from eicosapentaenoic acid. As conjunctival goblet cell secretion is critical for the health of the ocular surface, multiple different pathways are available to produce goblet cell secretion. If one pathway is blocked or depleted, others can potentially take over. Multiple, redundant pathways protect the cornea and ensure clear vision.
We conclude that goblet cells in the conjunctiva are able to respond to LXA 4 and other SPMS to maintain the homeostasis of the ocular surface. As LXA 4 directly affects goblet cells, lipoxins and other SPMs could be a novel treatment for dry eye diseases.

MATERIALS AND METHODS
Synthetic LXA 4 was purchased from EMD Millipore (Billerica, MA) and RvD1 was purchased from Cayman Chemical, Ann Arbor, MI). Both compounds were dissolved in ethanol as supplied by the manufacturer and were stored at −80 °C with minimal exposure to light. Immediately prior to use, each SPM was diluted in with Krebs-Ringer bicarbonate buffer with HEPES (KRB-HEPES, 119 mM NaCl, 4.8 mM KCl, 1.0 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 10 mM HEPES, and 5.5 mM glucose [pH 7.45]) to the desired concentrations and added to the cells. The LXA 4 analogs were prepared as described previously 16,17 . The cells were incubated at 37 °C in the dark. Daily working stock dilutions were discarded following each experiment. Annexin A1 (AnxA1) was purchased from MyBiosource (San Diego, CA). Aristolochic acid, 2-aminoethyl diphenylborinate (2-APB), 1 butanol and t-butanol were from Sigma Aldrich (St Louis, MO) while U0126 was purchased from R&D Systems (Minneapolis, MN). H89 and R0-31317549 were obtained from EMD Millipore (Billerica, MA).

Animals
Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing between 125 and 150 g were anesthetized with CO 2 for 1 min, decapitated, and the bulbar and forniceal conjunctival epithelia removed from both eyes. All experiments were in accordance with the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals and were approved by the Schepens Eye Research Institute Animal Care and Use Committee.

Cell Culture
Goblet cells from rat conjunctiva were grown in organ culture as described and extensively characterized previously 7,8,14,[43][44][45] . The tissue plug was removed after nodules of cells were observed. First passage goblet cells were used in all experiments. The identity of cultured cells was periodically checked by evaluating staining with antibody to cytokeratin 7 (detects goblet cell bodies) and the lectin Ulex europaeus agglutinin (UEA)-1 (detects goblet cell secretory product) to ensure that goblet cells predominated.

Reverse Transcriptase (RT)-PCR
Cultured goblet cells were homogenized in TRIzol and total RNA isolated according to manufacturer's instructions. One microgram of total RNA was used for complementary DNA (cDNA) synthesis using the Superscript First-Strand Synthesis system for RT-PCR (Invitrogen, Carlsbad, CA). The cDNA was amplified by the polymerase chain reaction (PCR) using primers specific to ALX/FPR2 receptor using the Jumpstart REDTaq Readymix Reaction Mix (Sigma-Aldrich, St. Louis, MO) in a thermal cycler (Master Cycler, Eppendorf, Hauppauge, NY). The primers for were from previously published sequences 9 . For rat goblet cells, the forward primer sequence was ATG GAA GCC AAC TAT TCC ATC, and the reverse primer sequence TCA TAT TGC TTT TAT ATC AAT GTT. These primers generated 1053 bp fragments. The conditions were as follows: 5 min at 95 ºC followed by 35 cycles of 1min at 94ºC, 30 s at annealing temperature for 1 min at 72 ºC with a final hold at 72 ºC for 10 min. Samples with no cDNA served as the negative control. Amplification products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.

Western blotting analyses
Cultured goblet cells were stimulated with increasing concentration of LXA4 and histamine (10-5 M) for 5 min. The reaction was stopped with the addition of excess ice cold KRB-HEPES and homogenized in RIPA buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA) in the presence of a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The homogenate was centrifuged at 2000g for 30 min at 4°C. Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and processed for western blotting. The antibody against the ALX/FPR2 receptor (Novus Biologics, Littleton, CO) was diluted 1:1000. Immunoreactive bands were visualized by the enhanced chemiluminescence method. The films were analyzed with Image J software (http://rsbweb.nih.gov/ij/).

Immunofluorescence Microscopy
First passage cells grown on glass cover slips and dissected rat conjunctiva were fixed in 4% formaldehyde diluted in phosphate buffered saline (PBS, 145 mM NaCl, 7.3 mM Na 2 HPO 4 , and 2.7 mM NaH 2 PO 4 (pH 7.2)) for 4 hours at 4ºC. Conjunctival tissue was preserved in 30% sucrose in PBS at 4ºC overnight and embedded in optimal cutting temperature embedding compound. Six micron sections were cut, placed on slides, and air dried for 2 hours. The sections or coverslips were rinsed for 5 minutes in PBS, and nonspecific sites were blocked by incubation with 1% bovine serum albumin, and 0.2% Triton X-100 in PBS for 45 minutes at room temperature. ALX/FPR2 receptor antibody (Novus Biologics) was used at 1:100 dilution overnight at 4 °C. Secondary antibodies were conjugated to Cy 3 (Jackson ImmunoResearch Laboratories, West Grove, PA) and were used at a dilution of 1:150 for 1 h at room temperature. Negative control experiments included incubation with the isotype control antibody. The cells were viewed by fluorescence microscopy (Eclipse E80i; Nikon, Tokyo, Japan) and micrographs were taken with a digital camera (Spot; Diagnostic Instruments, Inc, Sterling Heights, MI).

Measurement of Glycoconjugate Secretion
Cultured goblet cells were serum starved for 2 h before use, preincubated with inhibitors for 30 min, and then stimulated with either LXA 4 , histamine, or the cholinergic agonist carbachol (Cch) in serum-free RPMI 1640 supplemented with 0.5% BSA for 2 h. Goblet cell secretion was measured using an enzyme-linked lectin assay (ELLA) with the lectin UEA-I. UEA-1 detects high molecular weight glycoconjugates including mucins produced by rat goblet cells. The media were collected and analyzed for the amount of lectin-detectable glycoconjugates, which quantifies the amount of goblet cell secretion as described earlier 46 . Glycoconjugate secretion was expressed as fold increase over basal that was set to 1.

Measurement of [Ca 2+ ] i
Goblet cells were incubated for 1 h at 37 °C with KRB-HEPES plus 0.5% BSA containing 0.5 μM fura-2/AM (Invitrogen, Grand Island, NY), 8 μM pluronic acid F127, and 250 μM sulfinpyrazone followed by washing in KRB-HEPES containing sulfinpyrazone. Calcium measurements were made with a ratio imaging system (InCyt Im2; Intracellular Imaging, Cincinnati, OH) using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. At least 10 cells were selected in each experimental condition. After addition of agonists, data were collected in real time. Data are presented as the actual [Ca 2+ ] i with time or as the change in peak [Ca 2+ ] i . Change in peak [Ca 2+ ] i was calculated by subtracting the average of the basal value (no added agonist) from the peak [Ca 2+ ] i . Although data is not shown, the plateau [Ca 2+ ] i was affected similarly to the peak [Ca 2+ ] i .

Statistical analysis
Results were expressed as the fold-increase above basal. Results are presented as mean ± SEM. Data were analyzed by One-way ANOVA followed by tukey post hoc test or Student's t-test. P<0.05 was considered statistically significant.         . DAG activates PKC while IP 3 causes the release of Ca 2+ from intracellular stores. Extracellular regulated kinase 1/2 (ERK 1/2) is also activated. PLA2 causes the release of Ca 2+ . Intracellular Ca 2+ activates Ca 2+ /calmodulin kinase (Ca 2+ /CamK). All pathways lead to mucin secretion. Pathways in neutrophils are shown in B. LXA 4 or AnxA1 bind to ALX to activate PLD in a PKC dependent manner leading to shape changes and inhibition of granule mobilization A small amount of Ca 2+ is also released leading to increase in reactive oxygen species (ROS). Arachadonic acid (AA) is secreted. LXA 4 also activates c-Jun Nterminal kinase (JNK) which in turn activates caspase 3 leading to polymorphonuclear neutrophils (PMN) apoptosis.