Zoltan Fekete

Store-Operated Calcium Entry in Human Neutrophils Reflects Multiple Contributions from Independently Regulated Pathways

Human polymorphonuclear neutrophil (PMN) responses to G protein-coupled chemoattractants are highly dependent upon store-operated Ca2+ entry (SOCE). Recent research suggests that SOCE currents can be mediated by a variety of related channel proteins of the transient receptor potential superfamily. SOCE has been regarded as a specific response to depletion of cell calcium stores. We hypothesized that net SOCE might reflect the contributions of more than one calcium entry pathway. SOCE was studied in normal human PMN using Ca2+ and Sr2+ ions. We found that PMN SOCE depends on at least two divalent cation influx pathways. One of these was nonspecific and Sr2+ permeable; the other was Ca2+ specific. The two pathways show different degrees of dependence on store depletion by thapsigargin and ionomycin, and differential sensitivity to inhibition by 2-aminoethyoxydiphenyl borane and gadolinium. The inflammatory G protein-coupled chemoattractants fMLP, platelet-activating factor, and IL-8 elicit unique patterns of Sr2+ and Ca2+ influx channel activation, and SOCE responses to these agonists displayed differing degrees of linkage to prior Ca2+ store depletion. The mechanisms of PMN SOCE responses to G protein-coupled chemoattractants are physiologically diverse. They appear to reflect Ca2+ transport through a variety of channels that are independently regulated to varying degrees by store depletion and by G protein-coupled receptor activation.

Early trauma polymorphonuclear neutrophil responses to chemokines are associated with development of sepsis, pneumonia, and organ failure.

OBJECTIVES The modulation of polymorphonuclear neutrophil (PMN) function by injury is unpredictable, and can predispose either to hyperimmune states (adult respiratory distress syndrome [ARDS], multiple organ failure) or to immune dysfunction, infection, and sepsis. Such outcomes have been related to excess production of the CXC chemokine interleukin (IL)-8, but PMN responses to IL-8 are mediated by both the relatively stable and IL-8 specific CXC receptor 1 (CXCR1) and the labile, promiscuous CXCR2. We hypothesized that progression to septic and multiple organ failure outcomes could be related to early differences in PMN CXC receptor status. METHODS PMNs were isolated 12 +/- 3 hours after injury from 15 major trauma patients (Injury Severity Score of 34 +/- 2, 11 men and 4 women, age 36 +/- 4 years) who survived at least 7 days. Volunteer normal PMNs (n = 6 donors) were studied for comparison. Cells were stimulated either with the CXCR2 specific agent growth-related oncogene-alpha, or with IL-8, which stimulates CXCR1 and CXRR2. Receptor response was assessed as the mobilization of cell calcium. The development of ARDS, sepsis, and pneumonia was assessed according to standardized criteria. Day 1 receptor activity in the clinical groups was then compared by analysis of variance with Tukeys or t tests as appropriate. RESULTS In patients that were otherwise comparable, CXCR2 responses were markedly diminished in the PMNs of patients who went on to sepsis and pneumonia, but were elevated in PMNs from the patients who went on to ARDS. CXCR1 responses were modestly lower in trauma patients than volunteers, but showed no significant variations among the various clinical outcome groups. CONCLUSION The activity of PMN CXCR2 receptors soon after injury may be reflected in the later clinical sequelae of PMN activity. High CXCR2 activity may correlate with PMN hyperfunction and outcomes such as ARDS, whereas the loss of CXCR2 function in inflammatory environments may impair PMN functions in a manner that predisposes to pneumonia or sepsis. Early responses of PMN CXC receptors to injury may influence the clinical course of trauma patients.

Trauma-hemorrhage-induced neutrophil priming is prevented by mesenteric lymph duct ligation

Our objective in this study was to test the hypothesis that priming of neutrophils (PMN) in vivo by trauma-hemorrhagic shock (T/HS) is mediated by factors carried in intestinal lymph that prime PMNs by enhancing their responses to inflammatory mediators. Previous studies have shown that T/HS-induced lung injury is mediated by factors contained in mesenteric lymph and that ligation of the main mesenteric lymph duct (LDL) can prevent T/HS-induced lung injury. Since T/HS-induced lung injury is associated with PMN infiltration, one mechanism underlying this protective effect may be the prevention of PMN priming and activation. Therefore, we assessed the ability of T/HS to prime PMN responses to inflammatory agonists, and the ability of mesenteric lymph duct division to protect against such T/HS-induced PMN priming in an all-rat system. PMN were collected from male rats 6 h after laparotomy (trauma) plus hemorrhagic shock (30 mmHg for 90 min; T/HS) or trauma plus sham shock (T/SS). Uninstrumented rats were used as controls (UC). In a second set of experiments, rats were subjected to T/HS with or without mesenteric lymph duct division. PMN were then stimulated with chemokine (GRO, MIP-2) and lipid (PAF) chemoattractants, and cell calcium flux was used to quantify responses to those agonists. T/SS primed PMN responses to GRO, MIP-2, and PAF in comparison to UC rats, but the addition of shock (T/HS) amplified PMN priming in a significant manner, especially in response to GRO. Mesenteric lymph duct division prior to T/HS diminished PMN priming to the levels seen in T/SS. This reversal of priming was significant for GRO and GRO/MIP-2 given sequentially, with the other agonist regimens showing similar trends. The results support the concept that trauma and hemorrhagic shock play important additive roles in inflammatory PMN priming. Entry of gut-derived inflammatory products into the circulation via mesenteric lymph seems to play a dominant role in mediating the conversion of physiologic shock insults into immunoinflammatory PMN priming. Shock-induced gut lymph priming enhances PMN responses to many important chemoattractants, most notably the chemokines, and mesenteric lymph duct division effectively reverses such priming to priming levels seen in trauma without shock.

Shock mesenteric lymph-induced rat polymorphonuclear neutrophil activation and endothelial cell injury is mediated by aqueous factors.

BACKGROUND After trauma and hemorrhagic shock (T/HS), mesenteric lymph (ML) activates polymorphonuclear neutrophils (PMNs), injures endothelial cells (ECs), and predisposes to lung injury. The involved mediators, however, are unknown. We studied the ability of aqueous (AQ) and lipid (LIP) extracts of rat T/HS ML to activate PMNs and injure ECs. METHODS ML was collected from male rats undergoing trauma (laparotomy) plus hemorrhagic shock (30 mm Hg, 90 minutes) or sham shock. AQ and LIP ML fractions were separated using the Bligh-Dyer technique. Human umbilical vein endothelial cells were incubated 18 hours in 5% LIP or AQ lymph fractions and viability was assessed using the MTT assay. Rat PMNs incubated 5 minutes with 3% LIP or AQ fractions were assessed for respiratory burst (RB) and cytosolic calcium ([Ca(2+)](i)) using dihydrorhodamine 123 and fura-2AM. Human PMN responses to AQ and LIP T/HS lymph were studied similarly. RESULTS EC incubated in AQ showed 19 +/- 4% viability as compared with 65 +/- 11% in LIP (p < 0.001). Whole lymph affected ECs comparably to AQ T/HS lymph. Rat PMN basal [Ca(2+)](i) increased after exposure to AQ but not LIP T/HS lymph extracts. AQ T/HS lymph primed [Ca(2+)](i) responses to macrophage inflammatory protein-2 and platelet-activating factor; neither LIP T/HS nor any trauma and sham shock lymph fraction caused PMN priming. Rat PMN RB was elevated after AQ T/HS lymph incubation when compared with buffer (610 +/- 122 U/s vs. 225 +/- 38 U/s, p = 0.01). Rat PMN incubation in LIP T/HS lymph caused minimal activation (289 +/- 28 U/s, p = NS). Conversely, human PMN showed [Ca(2+)](i) and RB priming by rat T/HS LIP and not AQ extracts. CONCLUSION T/HS mesenteric lymph contains multiple biologically active mediators. Both AQ and LIP extracts of T/HS lymph are toxic to human umbilical vein endothelial cells, with AQ more active than LIP. Only AQ T/HS lymph activates rat PMNs, although LIP rat lymph extract activates human PMNs. These findings demonstrate the complex nature of gut lymph-derived biologic factors as well as species-specific differences on PMN and EC physiology. Therapies directed at any one specific molecule or mediator are therefore unlikely to be successful.

Attenuation of Shock-induced Acute Lung Injury by Sphingosine Kinase Inhibition

BACKGROUND Prolonged elevations of cytosolic calcium concentrations ([Ca2+]i) are required for optimal neutrophil (PMN) activation responses to G-Protein coupled chemoattractants. We recently showed that the coupling of endosomal Ca2+ store depletion to more prolonged entry of external Ca2+ depends on cellular conversion of sphingosine to sphingosine 1-phosphate (S1P) by sphingosine kinase (SK). We therefore hypothesized that inhibition of SK might inhibit PMN activation and thus ameliorate lung injury after trauma and hemorrhagic shock (T/HS). METHODS Chemotaxis (CTX) of human PMN was studied using modified Boyden chambers in the presence or absence of the selective SK inhibitor, SKI-2. After determining the concentration of SKI-2 that inhibited human PMN CTX by 50% (IC50) we subjected rats to T/HS (laparotomy, hemorrhage to 30-40 mm Hg x 90 minutes, 3 hours resuscitation). We then studied rat PMN CD11b expression using flow cytometry and lung injury using the Evans Blue dye technique in the presence of IC50 doses of SKI-2 or vehicle given in pretreatment at laparotomy. RESULTS Human PMN CTX was suppressed slightly more than 50% by 40 micromol/L SKI-2 (233 +/- 20 vs 103 +/- 12 x 10(3) cells/well, p < 0.001). Rat PMN expression of CD11b after T/HS was decreased from 352 +/- 30 to 232 +/- 7 MFU (p < 0.001) in the presence 30 micromol/L SKI-2. Lung permeability to Evans Blue was decreased from 9.5 +/- 2 to 4.1 +/- 0.7% (p = 0.036.). SKI-2 did not cause hemodynamic instability or alter resuscitation requirements. CONCLUSION Modulation of PMN Ca entry via SK inhibition inhibits PMN CTX in vitro, and inhibits CD11b expression in vivo without major effects on hemodynamics. These cellular changes were associated with amelioration of lung injury in vivo in a rat model of T/HS. These findings suggest that SK inhibition allows modulation of inflammation via control of [Ca2+]i without the cardiovascular compromise expected with Ca2+ channel blockade. SK inhibition therefore appears to be an important novel candidate therapy for inflammatory organ injury after shock.

The immunomodulatory effects of damage control abdominal packing on local and systemic neutrophil activity.

Background: Damage control laparotomy (DCL) with abdominal packing has become commonplace after major trauma, but the immune consequences of DCL are unknown. Methods: We collected 37 fluid samples from laparotomy pads (LPF) removed from 28 patients 1 hour to 7 days after DCL. Samples from eight patients who underwent serial packing were assayed for their mediator content and effects on neutrophil (PMN) function. Respiratory burst (RB) to N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate (PMA), as well as PMN calcium ([Ca 2+ ] i ) mobilization by GRO-a and platelet-activating factor were studied using dihydrorhodamine and fura-2-acetoxymethyl ester fluorescence. Brief exposure to 20% LPF (LPF20) modeled LPF acting on peritoneal PMNs and 2% LPF (LPF2) modeled the systemic effects on PMNs. Endotoxin (ETX), GRO-a, and leukotriene B4 were assayed by enzyme-linked immunosorbent assay. Data analysis was by analysis of variance with Dunns comparisons or the Mann-Whitney test when indicated. Results: LPF increased N-formyl-methionyl-leucyl-phenylalanine-induced RB from 0.4 ± 0.1 x 10 3 counts per second (control) to 0.7 ± 0.1 (LPF2) to 1.3 ± 0.3 (LPF20) (p < 0.05), with LPF2 increasingly active at later times after injury. PMA-elicited RB was primed only by LPF2 from < 24 hours. Both LPF2 and LPF20 markedly suppressed GRO-a [Ca 2+ ] i flux. Suppression by LPF2 was maximal at < 24 hours, abating after 48 hours. Suppression of GRO-a response was dose dependent: 150 ± 8 nmol/L in control PMNs, 97 ± 19 after LPF2, and 59 ± 4 after LPF20 (all p < 0.05). [Ca 2+ ] i flux after 1 nmol/L platelet-activating factor was only suppressed (from 181 ± 14 nmol/L to 149 ± 15 nmol/L,p < 0.05) by LPF20. LPF contained ETX, GRO-a, and leukotriene B4 at 10- to 20-fold plasma concentration in trauma patients. Conclusion: DCL results in peritoneal ETX and mediator accumulation even when cultures are sterile. LPF exposure primes PMN RB elicited by nonreceptor- (PMA) or receptor-coupled agonists that resist receptor desensitization. Conversely, LPF suppresses PMN responses to agonists that undergo receptor desensitization at high mediator concentrations. PMN dysfunction in such circumstances probably reflects a concomitant priming of some cell functions (e.g., RB) and desensitization of other (receptor-dependent) functions after an exposure to concentrated mediators. Peritoneal mediator production after DCL may be ETX driven, and may contribute to systemic inflammatory response syndrome. DCL trades early hemostasis for later inflammation. This should be considered in planning management strategies.

CXCR2 stimulation primes CXCR1 [Ca2+]i responses to IL-8 in human neutrophils.

Neutrophil (PMN) priming and subsequent responses to the IL-8 presented on pulmonary endothelial surfaces may be crucial determinants of the development of adult respiratory distress syndrome after injury. Elevated plasma ELR+ C-X-C chemokine (CXC) levels might contribute to PMN priming after trauma, but the role of CXCs in priming circulating PMNs is unstudied. We evaluated the interactions of IL-8 and GRO-alpha in priming human PMN calcium fluxes [Ca2+]i within circulatory environments. At physiologic concentrations, GRO-alpha primes PMN for IL-8 mediated [Ca2+]i mobilization, whereas IL-8 abolishes GRO-alpha responses. Repeated GRO-alpha exposures further enhance IL-8 responses. PMN priming for IL-8 responses in normal plasma was CXCR2 dependent. CXCR2 was more responsive than CXCR1 to low levels of IL-8, together suggesting that CXCR2 is the important CXC receptor at circulating (i.e., low) agonist concentrations. CXCR1 stimulation down-regulated CXCR2 surface expression, whereas CXCR2 stimulation upregulated CXCR1 expression. GRO-alpha/ CXCR2 signaling enhanced post-receptor IL-8 initiated PMN [Ca2+]i influx as well as efflux. Sufficient stimulation of the CXCR1 terminated this cooperative relationship by downregulating surface expression of CXCR2. This study is the first to report that at physiologic concentrations, C-X-C chemokines can act on circulating human PMNs as an integrated system where CXCR2 agonists, rather than cross-desensitizing CXCR1, act to enhance signaling of IL-8 at CXCR1 both by receptor and post-receptor mechanisms. Such CXCR2 mediated priming of CXCR1/ IL-8 interaction may enhance PMN attack on the lung after injury.

Store-operated calcium channel inhibition attenuates neutrophil function and postshock acute lung injury

Background:A wide variety of neutrophil (PMN) functions are regulated by cytosolic calcium concentration. Calcium channel blockade might therefore decrease postshock inflammation but could also limit important cardiovascular compensations. PMN Ca2+ entry occurs, however, through store-operated calci

Major trauma enhances store-operated calcium influx in human neutrophils.

PURPOSE Chemotaxins from inflammatory sites prime or activate neutrophils (PMN) by using cytosolic calcium ([Ca2+]i) fluxes as second messengers. [Ca2+]i can be mobilized rapidly by receptor-mediated entry or store-release, or more slowly by store-operated calcium influx (SOCI). We studied [Ca2+]i mobilization by chemotaxins and how trauma impacts the calcium entry mechanisms used by chemotaxins. METHODS [Ca2+]i flux was studied by spectrofluorometry. The contributions of early and late [Ca2+]i currents to net calcium flux were compared after stimulation by more potent (fMLP, C5a, PAF) or less potent (IL-8, GRO-alpha, and LTB4) agonists. Store operated [Ca2+]i mobilization was reflected by the ratio of area under the [Ca2+]i efflux curve to peak [Ca2+]i (efflux curve). PMN from trauma patients (ISS > 25) and pair-matched volunteer (n = 7 pairs) were then primed and stimulated with thapsigargin to compare cell calcium stores and SOCI. RESULTS Late [Ca2+]i mobilization made more important contributions to fMLP, PAF, and C5a signals than to IL-8, GRO-alpha, or LTB4 (p < 0.01 all comparisons). Calcium stores and store release were only marginally lower after injury (p = not significant), but trauma PMN showed far higher [Ca2+]i influx after thapsigargin (p = 0.007), and greater net SOCI (p = 0.034). CONCLUSIONS SOCI may play an important role in PMN activation, and trauma increases PMN SOCI. Prolonged elevations of [Ca2+]i due to enhanced SOCI may alter stimulus-response coupling to chemotaxins and contribute to PMN dysfunction after injury.

Increased Calcium Stores in Platelets From African Americans

Differences in cation transport have been observed between African Americans and whites. These differences may underlie the increased predisposition of African Americans to essential hypertension. To further explore these racial differences, we used platelets as a cellular model for calcium regulation. We measured 45Ca fluxes in platelets from 21 African American and 25 white men. Additionally, using fura 2, we measured cytosolic free calcium levels in resting platelets and platelets treated with ouabain and thrombin. Platelet 45Ca uptake was described by two exchangeable pools: a small, rapidly exchangeable pool and a larger, slowly exchangeable pool. Both pools were larger in platelets from African Americans than from whites (263 versus 185 pmol per 1 x 10(8) platelets for the rapidly exchangeable pool, P < .05; 744 versus 532 pmol per 1 x 10(8) platelets for the slowly exchangeable pool, P < .01). 45Ca washout was described by a rapidly exchangeable pool and a static pool. The former was also higher in platelets from African Americans than from whites (246 versus 202 pmol per 1 x 10(8) platelets, P < .01). The cytosolic free calcium concentrations in resting platelets were lower in African Americans than in whites. After treatment with ouabain and thrombin, the sustained posttransient levels of cytosolic free calcium increased to a greater extent in platelets from African Americans (46.7 nmol/L) than from whites (34.5 nmol/L, P = .033). Platelets from African Americans demonstrate higher intracellular calcium stores than platelets from whites. This racial difference could explain the sensitivity of African Americans to vasoactive agents acting through calcium mobilization from intracellular stores and cytosolic calcium.