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Arachidonic acid (AA, and its leukotriene derivatives e.g.: LTB4) is an inflammatory mediator in post-shock mesenteric lymph that appears to act as an agonist on G-protein coupled receptors (GPCRs). These mediators prime neutrophils (PMNs) for an increased production of superoxide, implicated in the development of ALI. Hypertonic saline (HTS) has also been shown to have immunomodulatory effects such as attenuation of PMN priming by precluding appropriate clathrin-mediated endocytosis of activated GPCRs, thereby potentially attenuating ALI. We hypothesize that HTS inhibits priming of the PMN oxidase by these lipid mediators.
After PMNs were isolated from healthy donors, incubation was done in either isotonic buffer (control) or HTS (180 mmol/L) for 5 minutes at 37°C. The PMNs were then primed for 10 minutes with AA [5 μM] or 5 minutes with LTB4 [1 μM] and the oxidase was activated with 200 ng/ml of phorbol 12-myristate 13-acetate (PMA), a non-GPCR activator, and superoxide anion generation was measured via reduction of cytochrome c.
Both AA [5 μM] and LTB4 [1 μM] significantly primed the PMA activated respiratory burst (p<0.05, ANOVA, Newman-Keuls, n=4). HTS inhibited both AA and LTB4 priming of the respiratory burst.
These data indicate that HTS reduces the cytotoxicity of PMNs stimulated by these lipid mediators in vitro and further support the immunomodulatory effects of HTS.
Arachidonic acid (AA) is a 20-carbon, ω-6 polyunsaturated fatty acid that is a precursor to leukotrienes and other important mediators of inflammation . Both leukotriene B4 (LTB4) and AA prime human neutrophils (PMNs) for a subsequent increased superoxide production . Hypertonic saline (HTS) is a known inhibitor of clathrin-mediated endocytosis (CME) , a process crucial in the internalization of G-protein coupled receptors (GPCRs), which are seven transmembrane proteins that form the largest family of signal transducing membrane receptors . Moreover, HTS inhibits priming of PMNs for numerous priming agents, such as platelet activating factor (PAF) that induce pro-inflammatory changes in PMNs through activation of CME .
LTB4, an effective PMN chemoattractant and priming agent, signals through B leukotriene receptor 1 (BLT1), a GPCR . Although a number of reports have postulated that AA primes PMNs through a GPCR, the data is inconclusive [8–12]. We hypothesize that HTS, a CME antagonist, inhibits the human PMN priming activity of LTB4 and AA.
All reagents, unless otherwise specified, were purchased from Sigma Chemical Company (St. Louis, MO). Solutions were made from sterile water for injection, United States Pharmacopeia (USP), from Baxter Healthcare Corp. (Deerfield, IL). All buffers were made from the following stock USP solutions: 10% CaCl2, 23.4% NaCl, 50% MgSO4 (American Reagent Laboratories, Inc., Shirley, NY), sodium phosphates (278 mg/ml monobasic and 142 mg/ml dibasic), and 50% dextrose (Abbott Laboratories, North Chicago, IL). Furthermore, all solutions were sterile-filtered with Nalgene™ MF75 series disposable sterilization filter units purchased from Fisher Scientific Corp. (Pittsburgh, PA). Ficoll-Paque was purchased from GE Healthcare Biosciences (Piscataway, NJ). LTB4 and AA were purchased from Cayman Chemical (Ann Arbor, MI). The calcium binding fluorometric dye Indo-1 AM was purchased from Invitrogen Corporation (Grand Island, NY).
PMNs were isolated by standard techniques . Heparinized whole blood was drawn from healthy human donors after obtaining informed consent employing a protocol approved by the Colorado Multiple Institutional Review Board and Human Subjects Committee at the University of Colorado School of Medicine. These donors satisfied the questions pertaining to health for all blood donors as mandated by the FDA and the industry standards for all blood banks in the United States. PMNs were isolated by dextran sedimentation, ficoll-paque gradient centrifugation, and hypotonic lysis as previously described . Cells were resuspended to a concentration of 2.5 × 107 cells/ml in Krebs-Ringers-phosphate buffer with 2% dextrose (KRPD) (pH 7.35) and used immediately for all subsequent manipulations
After PMNs were isolated from healthy donors, incubation of neutrophils (3.75 × 105 cell) was done in either isotonic buffer at a Na+ concentration of 135mmol/L (control), or HTS at a Na+ concentration of 180mmol/L,for 5 minutes at 37°C. PMNs were then primed for 10 minutes with AA [5 μM] or 5 minutes with LTB4 [100 nM, 1 μM, 10 μM] and the oxidase was activated with 200 ng/ml of phorbol 12-myristate 13-acetate (PMA), a non-GPCR activator of the oxidase, and the maximal rate of superoxide anion generation was measured via reduction of cytochrome c at 550 nm in a Molecular Devices (Menlo Park, CA) microplate reader .
After loading with 5 μM of the fluorometric dye Indo-1 AM (Invitrogen, Grand Island, NY), PMNs were centrifuged and resuspensed in fresh, warm KRPD to a concentration of 1 ×x 106 cells/ml. PMNs were then incubated in either HTS or isotonic buffer, loaded into a Perkin-Elmer LS50B spectrofluorimeter (Norwalk, CT) with constant stirring, and separately stimulated with LTB4 and AA. Calcium concentrations were measured in real-time with excitation at 355 nm and dual emission wavelengths were monitored at 410 and 470 nm. Data was analyzed with the Grynkiewicz equation as previously described [14–16].
Statistical differences were determined by paired or independent ANOVA followed by the Newman-Keuls test for multiple comparisons based upon the equality of variance. Statistical significance was determined by p<0.05. All data are presented as the mean ± SEM.
In the isotonic buffer, AA at 5 μM primed PMNs such that, when activated by PMA, resulted in a Vmax of 1.29 ± 0.04 vs. 0.97 ± 0.07 nmol O2− /3.75 × 105 cells/min in the buffer-treated controls, which did not undergo AA priming. (Fig 1; n=4; p<0.05 ANOVA, Newman-Keuls). In HTS-incubated neutrophils, superoxide anion production for the AA-primed PMNs was 0.72 ± 0.07 vs 0.79 ± 0.16 nmol O2− /3.75 × 10 cells/min in the buffer-treated control (Fig 1; n=4; p<0.05 ANOVA, Newman-Keuls), which equated to a 100% inhibition when compared to its isotonic counterpart. HTS did not significantly inhibit buffer-treated controls when compared to identical PMNs under isotonic conditions.
Superoxide anion production for the 100 nM, 1 μM, and 10 μM LTB4 primed-neutrophils in the isotonic group had a Vmax of 3.56 ± 0.8, 3.9 ± 0.78, and 4.16 ± 0.84 nmol O2− /3.75 × 105 cells/min, respectively, vs 2.17 ± 0.19 nmol O2− /3.75 × 105 cells/min in the buffer-treated control (Fig 2; n=4; p<0.05). Superoxide anion production after activation by PMA in the HTS group for the 100 nM, 1 μM, and 10 μM LTB4 primed-human neutrophils were 2.4 ± 0.77, 2.69 ± 0.8, and 2.63 ± 1.07 nmol O2− /3.75 × 105 cells/min, respectively (Fig 2; n=4; p<0.05). This corresponds to a Vmax inhibition of 72% ± 22%, 58% ± 13%, and 78% ± 28% in the 100 nM, 1 μM, and 10 μM LTB4 primed-human neutrophils, respectively.
Intracellular calcium ion concentration, as seen in figures 3 and and4,4, shows a sudden spike as a result of AA or LTB4 addition. This calcium flux was a qualitative test used to ensure the activation of GPCR by its corresponding ligand and subsequent heterotrimeric G protein signaling that is crucial for the release of Ca2+ from the calciosomes. Therefore, as a result of LTB4 addition to PMNs, an increase in intracellular Ca2+ signifies ligand activation of its BLT receptors and subsequent release of G proteins to increase intracellular Ca2+ . Whether in isotonic or HTS conditions, the receptor-mediated increases in cytosolic Ca2+ is unaffected, and that HTS inhibition of the superoxide anion assay is likely downstream of the release of G proteins. Increase in intracellular calcium following AA stimulation suggests activation of a GPCR receptor and subsequent release of G proteins to increase intracellular Ca2+.
Multiple Organ Failure (MOF) remains the leading cause of in-hospital mortality following trauma/hemorrhagic shock (T/HS) and lipid mediators in the post hemorrhagic shock lymph (PHSML) have been implicated in its development . The conduit for these pro-inflammatory agents are the mesenteric lymphatics  . PHSML contains significant amounts of AA and lymph flow increases three-fold following T/HS and has been linked to the pathogenesis of ALI following gut ischemia reperfusion . AA, which is in the PHSML, primes PMNs, the effector cell in MOF . Thus, research has focused on decreasing the pro-inflammatory potential of PMNs following T/HS, particularly by HTS. With data showing that isotonic saline may actually cause increased neutrophil adhesion molecules and activation , coupled with in vitro evidence that HTS favorably dampens PMN cytotoxicity , there is ongoing research on the use of HTS as a potential resuscitation fluid of choice following T/HS. Our data presented here shows that HTS inhibits LTB4 and AA priming of the PMN oxidase in vitro, from 58–100%. Since HTS is a CME antagonist, these data support the hypothesis that both AA and LTB4 induce CME upon ligation of their respective receptors, followed by priming of the neutrophil oxidase. Our previous work with PAF showed that HTS also inhibited the neutrophil. Identical to previous data using PAF, HTS had no effect on the LTB4 and AA mediated increases in cytosolic Ca2+. These data indicate that the inhibition of AA and LTB4 priming did not affect ligand:receptor avidity and this increase in cytosolic Ca2+ is likely the result of the release of a Gα subunit which, in turn, release intracellular Ca2+from the calciosomes . Of note, HTS-incubated PMNs had a higher calcium concentration in either LTB4 or AA primed neutrophils. The exact reason for this occurrence is not entirely understood, however we believe it is because a defect in proper clathrin formation allows for a longer stimulated receptor, and therefore more dissociation of G-proteins followed by subsequent increase in calcium, before the receptor is completely desensitized. Whether this plays a role in increasing the cytotoxicity of neutrophils after they're activated remains to be proven. As a qualitative test, however, it confirmed our belief that the receptor:ligand interaction was not inhibited by presence of HTS. The full mechanistic detail of neutrophil metabolism of exogenous AA is currently unknown , although, AA has been reported to bind to the 5-oxo-ETE receptor , a known GPCR linked to increases in cytosolic Ca2+ upon ligand occupancy . AA has also been shown to activate the arachidonate-regulated calcium (ARC) channel, a calcium-selective cation channel , although to date neutrophils have no known ARC channels. Increases in intracellular calcium is necessary for activation of 5-Lipoxygenase, the enzyme responsible for the synthesis of leukotrienes from AA, further propagating a pro-inflammatory state [28, 29]. Since the lymph flow is increased threefold in PHSML, thereby increasing the amount of AA delivered into the circulation, AA may represent an important mediator in the development of ALI post T/HS.
Our previous work has shown that HTS inhibits superoxide production of neutrophils primed by PAF, another lipid mediator of acute inflammation, via inhibition of CME [30, 31]. The data presented here further supports the immunomodulatory role of HTS. In concert with HTS administration regarding timing , neutrophils were preincubated with either HTS or buffer for 5 minutes, which was the optimal time for HTS to have an effect on PMNs. Regardless of the percentage of HTS used, in vitro concentration of Na+ was 180 mmol/L for the HTS group, which was the achievable Na+ concentration in swine hemorrhagic/shock models.
In summary, we have shown that HTS inhibits LTB4 and AA priming of the PMN oxidase in vitro, and this inhibition suggests that AA primes human PMNs via activation of a GPCR. Future work is needed to elucidate the mechanisms of AA-priming in PMNs via known GPCRs, including: BLT1, BLT2, and 5-oxo-ETE receptors. Discovery of these signal transduction pathways may allow for pharmacological intervention to attenuate or preclude the proinflammatory activation of PMNs by these lipids and ultimately decrease the incidence and/or the morbidity and mortality of MOF in injured patients.
I would like to thank Sanchayita Mitra, Roopali Shah, and Fabia Gamboni for their availability and assistance.
Grants This work was made possible by the T32 GM-0008315 and P50 GM-49222 National Institutes of Health grants.
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*Presented at the Academic Surgical Congress on February 3rd, 2011.