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The Journal of Immunology, 2005, 175: 4981-4989.
Copyright © 2005 by The American Association of Immunologists

Mechanisms of Hypotonicity-Induced Calcium Signaling and Integrin Activation by Arachidonic Acid-Derived Inflammatory Mediators in B Cells1

Peimin Zhu, Xiaohong Liu, Edward F. Labelle and Bruce D. Freedman2

Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We previously characterized the initial steps in the activation of novel (calcium-permeant) nonselective cation channels (NSCCs) and calcium release-activated calcium channels in primary murine B lymphocytes. Phospholipase C products, namely diacylglycerol and D-myo-inositol 1,4,5-trisphosphate, were identified as proximal intracellular agonists of these respective channels following mechanical stimulation of B cells. However, neither the distal steps in NSCC activation nor the contribution of these channels to sustained mechanical signaling were defined in these previous studies. In this study, single cell measurements of intracellular Ca2+ were used to define the mechanisms of NSCC activation and demonstrate a requirement for arachidonic acid liberated from diacylglycerol. Several arachidonic acid-derived derivatives were identified that trigger Ca2+ entry into B cells, including the lipoxygenase product 5-hydroperoxyeicosatetranenoic acid and the cytochrome P450 hydroxylase product 20-hydroxyeicosatetraenoic; however, the cytochrome P450 epoxygenase product 5,6-epoxyeicosatrienoic acid is primarily responsible for hypotonicity-induced responses. In addition to regulating calcium entry, our data suggest that eicosanoid-activated NSCCs have a separate and direct role in regulating the avidity of integrins on B cells for extracellular matrix proteins, including ICAM-1 and VCAM-1. Thus, in addition to defining a novel osmotically activated signal transduction pathway in B cells, our results have broad implications for understanding how inflammatory mediators dynamically and rapidly regulate B cell adhesion and trafficking.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Intracellular calcium has been implicated in nearly every function and fate of lymphocytes. Data suggest that modulations in the amplitude and frequency of cytoplasmic calcium levels dictate the pattern of transcription factor activation and cell differentiation (1, 2, 3, 4, 5). However, critical aspects of the mechanisms that regulate these distinct signals are still unknown. It is well established that Ag receptor-linked tyrosine kinases activate phospholipase C (PLC),3 which catalyzes the hydrolysis of membrane phosphatidyl-D-myo-inositol-4,5-bisphosphate to D-myo-inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 activates receptor/channels embedded within internal membranes, presumed to be the endoplasmic reticulum, allowing calcium to move down its concentration gradient into the cytoplasm. Distinct channels termed calcium release-activated calcium (CRAC) channels within the plasma membrane are activated in response to depletion of endoplasmic reticulum calcium stores (for review, see Ref.6). Although this axis between the Ag receptor, PLC activation, IP3-induced release of calcium from stores, and CRAC channel activation is assumed to underlie most, if not all, of the numerous Ca2+-dependent activities of lymphocytes, for most functions this has not been demonstrated.

Until recently, little attention has focused on other mechanisms of calcium signaling, including the role of DAG-regulated pathways. In B cells, DAG activation of protein kinase C (PKC) {beta} negatively regulates Ag receptor-mediated calcium signaling by blocking localization of the tyrosine kinase Btk to the Ag receptor complex (7); however, positive regulation of calcium entry by DAG in primary B cells was only recently described (8). In these previous studies, we demonstrated that mechanical forces, including those generated by hypotonicity-induced cell swelling, activate PLC, which produces DAG involved in nonselective cation channel (NSCC) activation (8).

Efforts detailed in this work focus on important questions raised by our initial studies regarding the distal mechanisms of NSCC activation and the contribution of these pathways to sustained Ca2+ signaling, as well as calcium-independent functions in B cells. We demonstrate that arachidonic acid (AA) liberated from DAG by DAG lipase is central to mechanically induced NSCC channel activation in B cells. AA is a precursor of several families of inflammatory mediators (eicosanoids), including leukotrienes, and oxilipids generated by lipoxygenase or cytochrome P450 (CYP450) enzymes. In B cells, lipoxygenase and CYP450 products of AA each trigger calcium entry; however, our data suggest that the CYP450 epoxygenase product 5,6-epoxyeicosatrienoic acid (5,6-EET) is principally responsible for hypotonicity-induced responses. These responses include an elevation in cytoplasmic Ca2+ concentration, but also adhesion to ICAM-1 and VCAM-1 due to NSCC-mediated changes in membrane potential (Vm) (8). Because hypotonicity and fluid shear force activate similar, if not identical, NSCCs in B cells, we have used hypotonicity as a means of inducing strain in the plasma membrane to mimic forces that B cells might encounter in the vasculature and lymphatics. We hypothesize that (eicosanoid) proinflammatory mediators produced in response to these mechanical stimuli regulate B cell trafficking by activating NSCCs and inducing B cell adhesion. Thus, we have identified a novel mechanism of signaling in B cells with broad implications for understanding how AA-derived inflammatory mediators, produced by a wide range of stimuli, regulate intracellular Ca2+ and B cell adhesion to extracellular matrix proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Intracellular calcium measurements

Splenic B lymphocytes were purified by immunomagnetic depletion, as previously described (8). Purified B cells were loaded with the cell-permeant calcium indicator fura 2-AM (3.0 µM; Molecular Probes) in RPMI 1640 medium for 15 min at room temperature (25°C). Cell suspensions were placed into a recording chamber on an inverted fluorescence microscope (Nikon) and allowed to adhere to poly-L-lysine (100 µg/ml; Sigma-Aldrich)-treated coverslips for 5 min in a solution that contained 150 mM NaCl, 4.5 KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (300 mOsm; pH 7.4). Excess fura 2-AM was removed by perfusing the chamber with extracellular solution. Intracellular Ca2+ was measured by digital imaging microscopy, as previously described (8). All results are expressed as the fura 2 fluorescence emission ratio at 510 nm obtained from cells sequentially excited at 340 and 380 nm. Calcium-containing hypotonic solutions contained 92 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4; 200 mOsm). Isotonic Ca2+-free bath solution contained 150 mM NaCl, 4.5 mM KCl, 2 mM MgCl2, 0.25 mM EGTA, 10 mM glucose, and 10 mM HEPES (pH 7.4; 300 mOsm), and hypotonic Ca2+-free bath contained 92 mM NaCl, 4.5 mM KCl, 2 mM MgCl2, 0.25 mM EGTA, 10 mM glucose, and 10 mM HEPES (pH 7.4; 200 mOsm).

AA, phorbol-12,13-didecanoate, PMA, DAG kinase (DGK) inhibitor II, and RHC-80267 were obtained from EMD Biosciences. The 20-hydroxyeicosatetraenoic (20-HETE), 5-hydroperoxyeicosatetranenoic acid (5-HPETE), acetylsalicylic acid (aspirin), nordihydroguaiaretic acid (NDGA), 5,8,11,14 eicosatetraynoic acid (ETYA), and 17-octadecynoic acid (17-ODYA) came from BIOMOL Research Laboratories. The membrane-permeant DAG 1-oleoyl-2-acetyl-sn-glycerol (OAG), fatty acid amide hydrolase (FAAH), miconazole (mico), 2,4'-dibromoacetophenone, and bromoenol lactone (BEL) were obtained from Sigma-Aldrich. The BCR was stimulated using soluble anti-mouse IgM F(ab')2 Ab (Jackson ImmunoResearch Laboratories).

Electrophysiology

Patch clamp measurements were performed on purified murine B cells (see above), or splenocytes identified visually by negative immunofluorescence (CD4 and CD8 negative) in the microscope recording chamber, as previously described (8). Briefly, patch pipettes were fabricated with a 4–6 M{Omega} tip resistance (Sutter Instruments) from borosilicate glass and were back-filled with appropriate internal solution. Liquid junction potentials were calculated and were corrected manually with the patch clamp amplifier or postanalysis. Following formation of gigaohm seals (5–10 G{Omega}), cells were lifted off the chamber bottom and held at –70 mV. Command potentials were generated using an EPC-9 patch clamp amplifier, and currents acquired, stored, and analyzed using PulseFit software (HEKA Electronics). The instantaneous current reversal potential was determined by applying 160 mV voltage ramps (–80 mV to +80 mV, 200 ms) to the patched cell before and after stimulation. Ramp currents measured after current activation were leak corrected by subtracting ramp currents obtained immediately after establishment of a stable whole cell recording. The standard extracellular bath solution used to measure currents contained 155 mM Na+ gluconate, 2 mM Ca2+ gluconate, 1 mM MgSO4, 10 mM Na+ HEPES, and 10 mM glucose adjusted to pH 7.2 with NaOH. The standard pipette solution contained [155C]methanesulfonate, 6 mM MgSO4, 1 mM EGTA, 0.25 mM calcium gluconate, and 10 mM HEPES (adjust pH to 7.3).

Inositol phosphate measurements

Purified B cells were suspended in RPMI 1640 (Invitrogen Life Technologies) containing 10% FBS, 2 mM glutamine, 1% oxaloacetate, pyruvate, and insulin (Invitrogen Life Technologies), and 1% nonessential amino acids at a concentration of 2 million/ml. Free [3H]inositol (2 µCi/ml; Amersham) was added to B cell cultures for 90 min at 37°C in a 5% CO2 incubator. LiCl (10 mM) was added for the last 30 min. Extracellular [3H]inositol was removed by washing cells once with fresh medium, and cells were allowed to rest for an additional 30 min. Cells were then stimulated for 60 s in the presence of LiCl (10 mM), and the response was stopped by adding an equal volume of 0.66 M cold (4°C) trichloracetic acid. Samples were centrifuged at 10,000 x g for 10 min at 4°C to pellet cellular and serum proteins, and supernatants were extracted four times with ether. The aqueous supernatants containing water-soluble inositol phosphates were applied to anion exchange columns (AG1-X8), and free inositol, IP1, IP2, and IP3 were eluted sequentially with water, 0.2 M, 0.4 M, and 0.8 M ammonium formate dissolved in 0.1 M formic acid. Eluted inositol phosphates were quantified by liquid scintillation counting.

AA release assay

Purified murine B cells (20 x 106 cells in 20 ml of B cell medium supplemented with 0.1% BSA) were labeled with 5 µCi of [3H]AA in 5% CO2 incubator at 37°C for 2 h. Cells were washed once and suspended (4 x 106/ml) in normal bath solution, treated with different blockers for 20–30 min, and then washed one time. Two million B cells in 1 ml of final volume were stimulated, as specified. Cells were then pelleted, and [3H]AA in supernatants was measured by scintillation counting.

Cell adhesion assays

Cell integrin avidity changes were assessed using an ICAM-1 and VCAM-1 adhesion assay similar to that previously described (8, 9). Immunolon 4 plates (Costar) were treated with anti-human Fc{gamma} (10 µg/ml in PBS; Jackson ImmunoResearch Laboratories) overnight at 37°C, and then wells were rinsed with PBS. Plates were blocked with 1% BSA in PBS for 1 h at 37°C to prevent nonspecific binding, after which plates were washed twice with PBS. Murine ICAM-1 (human) Fc fusion protein (10 µg/ml; R&D Systems), murine VCAM-1 (human) Fc fusion protein (20 µg/ml; R&D Systems), or human IgG (10 µg/ml; Jackson ImmunoResearch Laboratories) were added to wells for 1.5 h at 37°C, followed by two PBS washes. Splenocytes (4 x 106/ml, 100 µl) in complete RPMI 1640 and an additional 100 µl of complete medium containing indicated stimuli were added to each well. Cultures were incubated for 0.5 h at 37°C. Unbound cells and medium were removed by washing wells extensively with PBS. Adherent cells were subsequently removed from wells by incubating with complete RPMI 1640 containing 5 mM EDTA (200 µl) for 15 min at 4°C, enumerated, stained with anti-B220, and analyzed by flow cytometry to determine the absolute number of B cells recovered from each well. Results are expressed as the percentage of total B cells recovered from plates after subtraction of nonspecific binding values obtained from wells treated with human IgG. These studies were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mechanism of hypotonicity-induced Ca2+ mobilization in B cells

We previously reported that hypotonicity activates CRAC and NSCCs in B cells (8). Both of these are calcium permeant and could contribute to Ca2+ signaling. To further delineate the role of intracellular stores and these Ca2+ entry channels in hypotonicity-induced intracellular Ca2+ concentration ([Ca2+]i) changes, primary B cells were stimulated initially in Ca2+-free external medium to assess store release, and were subsequently superfused with Ca2+-containing solution to assess the contribution of Ca2+ entry to sustained signaling. Using this approach, we found that hypotonicity induces a transient [Ca2+]i elevation that decays to prestimulation levels after several minutes in Ca2+-free external solution, and a secondary sustained elevation following reintroduction of extracellular Ca2+ (Fig. 1A).



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  		FIGURE 1. Hypotonicity induces Ca2+ release from stores and IP3 production. A, Transient elevation in cytoplasmic Ca2+ is elicited in purified B cells by hypotonic (200 mOsm) Ca2+-free bath solution. Subsequent superfusion with Ca2+-containing hypotonic solution produces a secondary increase in cytoplasmic Ca2+ due to extracellular Ca2+ influx. Calcium was measured in single cells using fura 2 fluorescence emission ratio imaging, as described in Materials and Methods. B, Fold increase in inositol phosphate levels was measured in B cells within 60 s of stimulation. B cells loaded with [3H]inositol were placed in isotonic bath solution and subjected to hypotonicity (200 mOsm; left bars) or a concentration of anti-BCR Ab (40 µg/ml; right bars) found to induce maximal calcium signals. Tritiated IP1, IP2, and IP3 counts in cell lysates were quantified and normalized to corresponding levels in unstimulated control cells. Values represent mean ± SEM for four separate experiments. Analysis was performed using Student’s t test, and statistical comparison with controls are indicated (*, p = 0.04).

 
To better understand the regulation and contribution of Ca2+-permeant NSCCs to this response, we initially focused on their mechanism of DAG-dependent activation. We measured DAG levels in B cells before and after stimulation to assess any apparent concentration threshold for DAG-dependent Ca2+ mobilization. Hypotonicity induced a small, but inconsistent change in DAG levels, and following BCR engagement little, if any, measurable steady state increase (data not shown). These results suggested that either the rate of DAG metabolism is increased by the BCR and mechanical stimuli in proportion to any increase in production rate and/or that cellular localization of DAG, or enzymes that metabolize DAG, is more critical to its action than changes in total cellular steady state concentration, as demonstrated previously in T cells (10). Because we were unable to detect consistent changes in DAG concentration, we used an alternative strategy to assess its production. Given that PLC generates equal moles of IP3 and DAG from phosphatidyl-D-myo-inositol-4,5-bisphosphate, changes in inositol phosphate concentration (IP1, IP2, and IP3) should mirror the amount of DAG liberated. We found that BCR engagement produced a consistent increase (~1.2-fold, n = 4) in steady state IP3 and IP1 + IP2 levels within 60 s of stimulation, but that hypotonic stimulation induced a larger statistically significant (p = 0.04) elevation in IP3 (1.8-fold, n = 4) and its degradation products (IP1 + IP2 = ~1.6-fold) (Fig. 1B). These findings are consistent with our previous work demonstrating that hypotonic stress phosphorylates and activates PLC{gamma}-2 in B cells (8), but they also indicate that more IP3 (and vide infra DAG) accumulate following hypotonicity than BCR stimulation.

Mechanism of DAG-induced signaling

We next focused on the downstream mechanism by which DAG triggers Ca2+ entry in response to hypotonic challenge of B cells. DAG has a variety of effects in cells, many of which are mediated by PKC. To determine whether PKC plays a role in NSCC activation, we examined the effects of phorbol esters (phorbol-12,13-didecanoate or PMA) on [Ca2+]i. Neither agonist mobilized calcium (data not shown) nor elicited a transmembrane ion current in B cells (8), suggesting that PKC is not directly involved in NSCC activation by DAG. We next examined the role of DGK, which converts DAG to phosphatidic acid, because this enzyme has been shown to regulate T lymphocyte activation (11, 12). Hypotonicity-induced Ca2+ signals were normal in DGK{zeta}-deficient B cells (11) (data not shown) and in cells treated with the DGK ({alpha}) inhibitor II (data not shown). These results indicate that neither of the DGK isoforms identified in lymphocytes, or their products, are required for mechanical Ca2+ signaling in B cells.

DAG can also be degraded by the enzyme DAG lipase (Fig. 2A) to form AA. Consistent with a role for AA in NSCC activation, exogenous AA induces Ca2+ influx (Fig. 2B) and activates a nonselective cation current, which is biophysically indistinguishable from that activated by hypotonicity in B cells (Fig. 2C) (8) (data not shown). AA-induced currents, like those elicited by hypotonicity, develop slowly and often require several minutes to reach a peak steady state amplitude (37.3 ± 7.9 pA/pF; n = 15). However, in nearly half (47%) of all responding cells, AA-evoked currents exhibit biphasic activation kinetics (see Fig. 2C). In these cells, an initial small amplitude current (7.7 ± 1.2 pA/pF; n = 7) developed first, and were followed by a larger secondary increase in amplitude (–32.9 ± 6.6 pA/pF, Vm = –70 mV). Notably, the reversal potential for each of these components is indistinguishable (~0 mV; Fig. 2C, inset). Under the conditions of these measurements (see Materials and Methods), this indicates that AA activates at least one, but possibly multiple biophysically similar NSCCs in B cells.



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  		FIGURE 2. Mechanisms of AA metabolism and signaling. A, Signaling cascade showing mechanisms of AA production and metabolism. Metabolizing enzymes are shown in square boxes, precursors and products of AA are enclosed in ovals, and pharmacological inhibitors are enclosed in parentheses. Mico, miconazole; HETE, hydroxyeicosatetraenoic; HPETE, hydroperoxyeicosatetranenoic acid; EET, epoxyeicosatrienoic acid; aspirin, acetylsalicylic acid. B, AA (10 µM) does not mobilize intracellular Ca2+, but induces an increase in [Ca2+]i that is dependent upon extracellular Ca2+ influx. Calcium measurements were performed, as previously described. C, Patch clamp recording of current elicited by AA in B cells (holding potential = –70 mV). Approximately half exhibit a biphasic current similar to that shown, whereas the smaller initial current component was not observed in a small majority of recordings. The Erev (inset) of leak-subtracted currents measured at indicated time points (b = 0 mV; c = 2.5 mV) in normal (155 mM) extracellular Na+ are indicative of a nonselective cation permeability for each of these components (mean for all currents = –4.4 ± 3.4 mV). The inward current is blocked by RR (10 µM). D, AA release from B cells induced by hypotonic stress (200 mOsm) and BCR engagement (40 µg/ml). The response to hypotonicity is blocked by the DAG lipase inhibitor RHC-80269 (*, p = 0.03). RHC-80267 has no effect on BCR-induced AA release, which is less than that induced by hypotonicity (**, p = 0.07). Results shown represent the mean ± SEM of at least three independent trials for each condition. Statistical significance was determined using two-tailed Student’s t test.

 
AA appears to be a physiologically relevant mediator of Ca2+ mobilization, because hypotonic stimulation induces AA release from B cells (2.1-fold, n = 5, p = 0.03) and its production is fully blocked by the DAG lipase inhibitor RHC-80267 (Fig. 2D). By contrast, phospholipase A2 (PLA2) inhibitors (BEL, 2,4'-dibromoacetophenone, and chloroquine) did not exert any significant effect on hypotonicity-induced AA production (data not shown). Thus, AA produced by DAG lipase is primarily, if not exclusively, responsible for hypotonicity-induced Ca2+ entry in B cells. Interestingly, BCR engagement also induces AA release (Fig. 2D); however, the amount is less than that produced by hypotonic stress and its production is insensitive to the DAG lipase inhibitor RHC-80267. Thus, the BCR generates AA by a distinct mechanism than hypotonicity, possibly involving its liberation from membrane phospholipids by iPLA2 as recently demonstrated in basophils and smooth muscle cells (13). Consistent with such a mechanism, the iPLA2-selective inhibitor BEL does block BCR-induced store-dependent Ca2+ entry in B cells (see below). These differences in the source and quantity of AA produced by BCR engagement may account for our previous observation that BCR stimulation does not activate NSCCs (8).

Given that DAG lipase activity is required for AA production, we next examined the role of its precursors and products in hypotonicity-induced Ca2+ signaling (see Refs.14, 15, 16, 17). We found that DAG lipase inhibition did not affect Ca2+ store release, which was comparable to that in untreated cells (Fig. 3, A and B), but RHC-80267 almost completely abolished hypotonicity-induced Ca2+ influx (Fig. 3B). By contrast, PLA2 and FAAH inhibitors (see Fig. 2A) had no measurable effect on hypotonicity-induced signals (data not shown). Thus, DAG appears to be the primary source of AA for induction of calcium entry in B cells. It should be noted that in previous experiments we used OAG (a membrane-permeant DAG) to activate NSCCs. DAG lipase cleaves AA from the second side chain position on the glycerol backbone of endogenous cellular DAGs; however, OAG does not contain AA in this position. Nonetheless, AA, OAG, and hypotonicity activate membrane currents (Fig. 2C) (8) (data not shown), which are indistinguishable from one another. If the same channel is activated by each of these stimuli, then OAG must induce AA production via a distinct (DAG lipase-independent) mechanism. Indeed, OAG triggers AA release from B cells (Fig. 2C). Moreover, it has been demonstrated that DAG can directly activate PLA2 (18). Although we did not obtain evidence for physiological activation of PLA2 by hypotonicity in B cells, it is possible that pharmacological concentrations of DAG (i.e., 200 µM OAG) used in previous experiments and in the AA release assay above do so. Regardless of the mechanism of OAG action, AA generated from endogenous DAG is a critical intermediate linking hypotonic stimulation to Ca2+ signaling in B cells.



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  		FIGURE 3. Mechanism of hypotonicity-induced Ca2+ signaling. The role of AA precursors and products in hypotonicity-induced Ca2+ release from stores and extracellular influx. A, Typical transient elevation reflects Ca2+ release from intracellular stores, and secondary increase is due to influx of extracellular cation. Pretreatment with the DAG lipase inhibitor RHC-80267 (10 µM) (B) or ETYA (10 µM) (C), which is a broad inhibitor or AA metabolism, almost completely blocks hypotonicity-induced Ca2+ entry. D, The broad CYP450 inhibitor 17-ODYA (10 µM) produces a complete block of Ca2+ entry in the majority of B cells. E, The more selective CYP450 epoxygenase inhibitor miconazole (10 µM) produces a similar effect as 17-ODYA. F, The 17-ODYA (and miconazole)-resistant component of AA-induced calcium influx is further blocked by the lipoxygenase inhibitor NDGA (10 µM; see text for percentages). Results shown are typical of at least three separate trials for each treatment.

 
Eicosanoids produced from AA activate distinct Ca2+ entry pathways

AA is a precursor of numerous inflammatory mediators, including leukotrienes, PGs, prostacyclins, thromboxane, and other eicosanoids (e.g., HPETEs, HETEs, EETs; Fig. 2A). Given that hypotonicity induces AA release, we asked what role these eicosanoids, or the pathways involved in generating them, play in hypotonicity/AA-induced signaling in B cells. To address this question, we first examined the effect of inhibitors of eicosanoid production on hypotonicity-induced Ca2+ entry in B cells. Under control conditions, hypotonicity initiated Ca2+ entry in the majority of B cells (83.6 ± 2.0%, n = 3, >200 cells tested for each condition), and this response was blocked by the DAG lipase inhibitor RHC-80267 (Fig. 3B, 9.5 ± 2.3% responders). A general inhibitor of AA metabolism, namely ETYA, also blocked Ca2+ entry in the majority of cells (Fig. 3C; 14.1 ± 3.8% responders), consistent with a mechanical signal transduction pathway involving DAG and AA. Surprisingly, ETYA also partially inhibited Ca2+ release from stores. However, inhibitors that block steps further downstream than those affected by ETYA did not affect Ca2+ release from stores (see below).

We next determined which metabolites of AA mediate the response to hypotonicity. Inhibition of cyclo-oxygenases-1 and -2 with aspirin did not affect either AA- or hypotonicity-induced Ca2+ signals (data not shown), suggesting that PGs, prostacyclins, and thromboxane are not involved. Methanandamide, which inhibits AA production from endocannibanoids by FAAH (see Fig. 2A), also did not inhibit these responses. By contrast, the CYP450 hydroxylase/epoxygenase inhibitor 17-ODYA attenuated Ca2+ entry (Fig. 3D; 19.6 ± 5.1% responders, n = 3 experiments, >250 cells), as did the more selective CYP450 epoxygenase inhibitor miconazole (Fig. 3E; 27.8 ± 4.8% responders). The combination of miconazole and the 5-lipoxygenase inhibitor NDGA produced an additive effect (Fig. 3F; 15.1 ± 3.4% responders). Together, these data suggest that CYP450 epoxygenase activity and its products are principally responsible for hypotonicity-induced Ca2+ entry in B cells, but that 5-lipoxygenase and CYP450 hydroxylase products also may play a small role in this response.

These results are consistent with previous work demonstrating that the CYP450 epoxygenase product 5,6-EET is an agonist of calcium-permeant osmotically activated transient receptor potential V4 (TRPV4) NSCCs (17). We previously identified TRPV4 mRNA and TRPV4-like cation currents in primary B cells (8). Therefore, we tested the sensitivity of hypotonicity-activated Ca2+ signals to the TRPV4 blocker ruthenium red (RR) (18). RR did not inhibit hypotonicity-induced Ca2+ release from stores, but did attenuate Ca2+ influx (Fig. 4A). RR also blocked 5,6-EET-induced Ca2+ entry (Fig. 4B), but had no effect upon responses to products of 5-lipoxygenase (5-HPETE; Fig. 4C) or CYP450 hydroxylase (20-HETE; Fig. 4D). Together, these results demonstrate that several eicosanoids are capable of elevating Ca2+ in primary B cells, but that the predominant effect of hypotonicity is mediated by a 5,6-EET-activated RR-sensitive channel.



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  		FIGURE 4. Identification of eicosanoids involved in hypotonicity-induced Ca2+ entry. Multiple AA acid metabolites were tested for the ability to trigger Ca2+ release from stores and influx in primary B lymphocytes. Calcium release from stores and influx were assessed separately using the Ca2+-free-Ca2+ add back protocol previously described. A, Hypotonicity induced a transient elevation due to intracellular Ca2+ release from stores and a secondary sustained increase following reperfusion of cells with Ca2+-containing bath solution. The secondary influx-associated increase is attenuated by RR (10 µM; right panel). B, The CYP450 epoxygenase product of AA, 5,6-EET, does not mobilize intracellular Ca2+, but activates plasma membrane entry pathways. Ca2+ entry is blocked substantially by RR (right panel). C, The 5-lipoxygenase product, 5-HPETE, and D, the CYP450 hydroxylase product 20-HETE of AA also trigger Ca2+ entry in B cells, but neither of these increases is sensitive to RR (right panels).

 
Distinct mechanisms of hypotonicity- and BCR-induced Ca2+ signaling

Our pharmacological characterization of Ca2+ signals suggests that sustained hypotonic signaling is mediated primarily by a DAG-dependent entry mechanism (i.e., NSCCs); yet, previous patch clamp measurements demonstrated that osmotic stress also activates CRAC channels, which are Ca2+ store operated (8). This is not unexpected given the fact that hypotonicity induces significant Ca2+ release from intracellular stores (Figs. 1, 3, and 4). However, following inhibition of NSCCs (with inhibitors of DAG or AA metabolism; Fig. 3), relatively little (CRAC channel-mediated) Ca2+ entry was evident. This surprising result suggested that CRAC channels are activated by, but contribute relatively little to, hypotonicity-induced intracellular Ca2+ elevations. To validate this interpretation, it was first necessary to determine the effect of eicosanoid signaling inhibitors on the response to BCR engagement, which mobilizes intracellular stores and activates CRAC, but not NSCCs (8). As previously shown, BCR engagement produces a rapid increase in [Ca2+]i in control cells that decays to a sustained elevation (Fig. 5A), and neither ETYA (Fig. 5C) nor RR (Fig. 5D) has any significant effect on this response. RHC-80267 produces some suppression of BCR-induced Ca2+ release from stores and steady state Ca2+ levels (Fig. 5B); however, we believe that this effect of RHC-80267 is due to feedback inhibition by accumulated DAG, which can negatively regulate BCR-induced Ca2+ entry (7) (data not shown).



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  		FIGURE 5. Distinct mechanisms of BCR- and hypotonicity-induced Ca2+ entry. A, BCR engagement with soluble anti-IgM (40 µg/ml) triggers a biphasic increase in [Ca2+]i in purified primary B cells in normal isotonic extracellular solution. B, The DAG lipase inhibitor RHC-80267 modestly suppresses (~25%) the peak and steady state sustained Ca2+ levels. ETYA (C) and RR (D) have no significant effect on mean peak or sustained BCR-induced [Ca2+]i elevations.

 
Additional support for the distinct nature of BCR- and hypotonicity-induced Ca2+ entry mechanisms was obtained using the iPLA2 inhibitor BEL, which was recently shown to inhibit CRAC channel activation (13, 19). In contrast with NSCC inhibitors and consistent with these previous reports, BEL almost completely blocks BCR-induced (CRAC-mediated) Ca2+ entry in primary B cells (Fig. 6A). BEL has a small effect on the initial hypotonicity-induced increase in Ca2+, but had no discernible effect on sustained elevations produced by entry (Fig. 6B). In fact, BEL-resistant hypotonicity-induced Ca2+ elevations are similar to those produced in B cells by direct application of eicosanoids (Fig. 4), which do not trigger an initial Ca2+ spike associated with Ca2+ release from stores. Thus, BEL appears to selectively block the initial contribution of store release and CRAC channel activation to hypotonicity-induced responses. Together, these differences in pharmacological sensitivity of BCR- and hypotonicity-induced Ca2+ entry demonstrate that distinct physiological mechanisms underlie each response.



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  		FIGURE 6. Differential role for PLA2 in BCR- and hypotonicity-induced Ca2+ signaling. B cells were stimulated in the absence (left panels) or presence (right panels) of the PLA2 inhibitor BEL in normal bath solution. A, In the presence of BEL, BCR engagement produces a small transient elevation in Ca2+ due to release from stores, but no apparent entry of extracellular ion. B, Hypotonicity induces a typical biphasic increase in Ca2+ (left) in the absence of inhibitor. BEL inhibits the initial transient increase in Ca2+, but has no discernible effect on sustained signaling (right). These results are typical of at least three different experiments with each condition.

 
Direct activation of integrins by AA signaling

We previously demonstrated that hypotonicity and OAG increase the avidity of B cell integrins for extracellular matrix proteins ICAM-1 and VCAM-1. Integrins play an important role in lymphocyte trafficking to sites of inflammation and in stabilizing interactions with other cells of the immune system. Our previous results also suggested that NSCCs themselves, by virtue of their ability to regulate the plasma membrane potential, and not other DAG-dependent pathways, are responsible for this integrin avidity modulation (8). Consequently, if signal transduction intermediates identified downstream of endogenous DAG (i.e., AA and 5,6-EET) are capable of eliciting similar responses as hypotonicity and DAG, this would support a direct role for eicosanoid-regulated channels in integrin avidity modulation. To assess this, we measured the effect of AA and 5,6-EET on B cell adhesion to ICAM-1 and VCAM-1. Consistent with our previous work (8), hypotonicity increased B cell adhesion to VCAM-1 (Fig. 7A) and ICAM-1 (Fig. 7B), and this induction was inhibited by RHC-80267, ETYA, and RR. AA elicited a similar increase in VCAM-1 and ICAM-1 binding as hypotonicity and these increases were attenuated by ETYA and RR. The proximal TRPV4 agonist 5,6-EET also increased binding by a RR-sensitive mechanism. These results demonstrate that modulation of ICAM-1 and VCAM-1 binding by hypotonicity involves the same molecules that activate NSCCs in B cells. With respect to ICAM-1 binding, although each of the drugs consistently inhibited binding, the effects were not statistically significant (i.e., p > 0.05). The lack of statistical significance in these experiments may reflect differences in the regulation of binding to these two adhesion molecules (i.e., less binding to ICAM-1) and the greater variability seen for the induction of ICAM-1 binding. Nonetheless, these results also suggest that NSCC activation is an important mechanism of integrin activation that occurs in response to production of AA from DAG specifically, but also could occur in response to AA produced from substrates other than DAG.



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  		FIGURE 7. Integrin activation by AA and 5,6-EET. B lymphocytes were subjected to hypotonicity (200 mOsm) or treated with AA (10 µM) or 5,6-EET (10 µM) to define the mechanism of VCAM-1 (A) and ICAM-1 (B) binding. Hypo-osmotic stimulation of B cells induces an increase in VCAM-1 (p = 0.01) binding. RHC-80267 (10 µM), ETYA (10 µM), and the TRPV channel inhibitor RR (10 µM) each block this increase by at least 75%. Although the increase in ICAM-1 binding induced by hypotonicity in these experiments is less significant (p = 0.09) than VCAM-1 binding, each is blocked by inhibitors of AA metabolism (ETYA) and TRPV channels (RR). AA (10 mM) also induces a significant increase in VCAM-1 (p < 0.01) and a less significant increase in ICAM-1 binding (p = 0.09). The TRPV4 agonist 5,6-EET increases both VCAM-1 (p = 0.04) and ICAM-1 binding by an RR-sensitive mechanism. Each value represents the mean ± SEM for at least three independent experiments. Statistical analysis was performed using paired Student’s t test (*, significant increase at confidence; p ≤ 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previously, we reported that fluid shear force and osmotic (hypotonic) stress mobilize calcium in primary B lymphocytes. These stimuli, like those produced by BCR engagement, activate PLC, which releases Ca2+ from IP3-sensitive intracellular stores, thereby activating CRAC channels. However, hypotonic stress (but not the BCR) also activates (DAG-dependent) Ca2+-permeant NSCCs (8). Therefore, a primary objective of this study was to define the mechanism of mechanically induced Ca2+ signaling in B cells and specifically the role of CRAC vs nonselective cation channels. By measuring the production of key mediators of CRAC and NSCC activation and intracellular Ca2+ in single B cells, we have identified a novel signal transduction pathway that links DAG to (NSCC-mediated) calcium entry and integrin activation. Our results demonstrate that AA is produced from DAG by DAG lipase. Multiple AA-derived mediators were found to be capable of subsequently triggering Ca2+ entry in B cells; however, a product of CYP450 epoxygenase, namely 5,6-EET, is primarily responsible for sustained hypotonicity-induced Ca2+ entry.

Our results also suggest that CRAC channels play a relatively minor role in sustained hypotonic signaling, which is surprising given our observation that hypotonicity produces a significant increase in steady state IP3 levels, in fact, greater than that induced by the BCR. This suggests that Ca2+ release from stores, or the mechanism by which store depletion activates CRAC channels, is negatively regulated by some element of mechanical signaling pathways. We had hypothesized that DAG required for NSCC activation might be targeted or localized by the BCR to channel inaccessible compartments within B cells; consequently, DAG levels in proximity to NSCCs would be insufficient to activate them (8). However, given our finding that the BCR generates less DAG and IP3, we now favor the hypothesis that the critical DAG concentration threshold for NSCC activation is higher than the molar concentration of IP3 required for full activation of CRAC channels, and the BCR does not exceed this DAG threshold. This does not explain why CRAC channel activation by hypotonicity (observed in the presence of RR, ETYA, or RHC-80267) produces smaller sustained Ca2+ elevations than does BCR engagement alone, but does demonstrate that CRAC channel activation is attenuated during the response to hypotonicity. The fact that hypotonicity produces more IP3 and presumably more DAG than the BCR suggests that DAG-dependent mechanisms regulate the contribution of CRAC channels to hypotonicity-induced responses. The attenuation appears to involve a step distal to store release, because store release was not affected by NSCC activation inhibitors.

Given the apparent requirement for PLA2 in CRAC, but not NSCC activation (13, 19) (Fig. 6), it is also possible that PLA2 or AA could be involved in hypotonicity-induced suppression of CRAC channel activation. Reciprocal regulation of store-operated and AA-regulated channels has been observed in a number of cell types (20, 21). However, these AA-regulated channels are biophysically and pharmacologically distinct from the AA-sensitive channels we have defined in B cells (22). For example, AA-regulated channels are significantly more Ca2+ selective and Na+ impermeant under physiological conditions, have a positive reversal potential, are insensitive to blockers such as 2-APB, which blocks AA-regulated NSCCs channels in B cells, and are directly activated by AA, but not eicosanoid products of AA. Moreover, we did not observe AA suppression of BCR or thapsigargin-induced Ca2+ signals in B cells; rather, we found that AA treatment enhances BCR signaling (data not shown). Consequently, a distinct mechanism is responsible for cross-regulation of store-operated calcium signaling by DAG/AA-regulated pathways in B cells than in these other systems. Nonetheless, the general paradigm that Ca2+ store-operated (CRAC) and store-independent AA-regulated (NSCC) channels produce distinct patterns of Ca2+ signaling and participate in discreet functional responses is consistent with our findings in B cells.

The pharmacological properties of hypotonicity-, AA-, and 5,6-EET-induced Ca2+ signals, including their sensitivity to RR inhibition, and biophysical properties of hypotonicity- and AA-activated currents (see Fig. 2) (8) resemble those of TRPV4 NSCCs (17). However, Ca2+ entry triggered by other eicosanoids, including 20-HETE and 5-HPETE, were insensitive to RR, suggesting that these oxilipids target different channels than 5,6-EET. In fact, 20-HETE can activate heterologously expressed TRPC6 (23), whose mRNA is also expressed in primary murine B cells (8). Direct genetic manipulation of these channels will be the only definitive way to determine their molecular identity, and this is the focus of ongoing patch clamp and molecular genetic studies.

The novel Ca2+-signaling mechanisms that we have identified in B cells have important functional implications. It has been established that Ca2+ is capable of triggering a wide range of distinct lymphocyte fates and functions (2, 5), but how this critical fate-specific information is encoded into distinct patterns of intracellular calcium signaling remains an open question. We demonstrate that AA derived from DAG couples hypotonic stimulation to Ca2+ entry and similar Ca2+-signaling elements can be activated independently of PLC, by pathways involved in AA metabolism (e.g., PLA2 or FAAH). AA-derived intermediates are thereby capable of mobilizing intracellular Ca2+, but, importantly, triggering functional responses such as integrin activation, independently of Ca2+ store release or CRAC channel activation. In fact, AA generated by PLA2 has been implicated in the generation of proinflammatory eicosanoids that regulate the production of cytokines, NO, and free radicals involved in the pathogenesis of immune-mediated inflammatory diseases such as multiple sclerosis (24, 25). Eicosanoids are also produced by some prokaryotic and eukaryotic pathogens (26) and induce inflammation and host susceptibility to these pathogens (9). In addition, eicosanoids produced by vascular endothelium increase vascular permeability and promote adhesion and transmigration of leukocytes (27). Thus, eicosanoids secreted by B and other host cells or by pathogens at sites of inflammation may mediate cross talk between them that promotes and coordinates the inflammatory response.

In summary, we have defined the steps by which hypotonicity mobilizes Ca2+ in B cells; however, our results also point to a new general mechanism by which a variety of eicosanoid inflammatory mediators in addition to leukotrienes, PGs, and prostacyclins, generated from AA, activate integrin-dependent adhesion. Data in this manuscript do not directly address the mechanism of integrin activation, including the role that Ca2+ may play, although previously we determined that NSCC-mediated changes in membrane potential and not Ca2+ signals are responsible for integrin activation. Regardless, the signal transduction pathways responsible for NSCC-mediated Ca2+ entry and membrane potential modulation represent a physical mechanism, distinct from Ag receptor-coupled pathways, capable of initiating fate-specific responses of B cells to a wide array of inflammatory mediators, produced by lymphocytes, vascular endothelium, pathogens, and other leukocytes at sites of inflammation. These important responses of B cells operate independently of intracellular Ca2+ stores and CRAC channels operated by the BCR, yet work in concert to coordinate the immune response.


   Acknowledgments
 
We thank Zhiyun Wen for her technical contributions.


   Disclosures
Top
 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 National Institutes of Health Grants AI 39678 and AI 49091 to B.D.F. Back

2 Address correspondence and reprint requests to Dr. Bruce D. Freedman, University of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology, Room 368E Old Vet Building, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: bruce{at}vet.upenn.edu Back

3 Abbreviations used in this paper: PLC, phospholipase C; AA, arachidonic acid; BEL, bromoenol lactone; [Ca2+]i, intracellular Ca2+ concentration; CRAC, calcium release-activated calcium; CYP450, cytochrome P450; DAG, diacylglycerol; DGK, DAG kinase; 5,6-EET, 5,6-epoxyeicosatrienoic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; FAAH, fatty acid amide hydrolase; 20-HETE, 20-hydroxyeicosatetraenoic; 5-HPETE, 5-hydroperoxyeicosatetranenoic acid; IP1, D-myo-inositol monophosphate; IP2, D-myo-inositol 1,4-bisphosphate; IP3, D-myo-inositol 1,4,5-trisphosphate; iPLA2, calcium-independent phospholipase A2; TRP, transient receptor potential; Vm, membrane potential; NDGA, nordihydroguaiaretic acid; NSCC, nonselective cation channel; OAG, 1-oleoyl-2-acetyl-sn-glycerol; 17-ODYA, 17-octadecynoic acid; PKC, protein kinase C; PLA2, phospholipase A2; RR, ruthenium red. Back

Received for publication March 4, 2005. Accepted for publication July 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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