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-α, 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-α, 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.
Injury elicits synthesis and release of a variety of chemotactic mediators of inflammation. Most chemotaxins alter neutrophil (PMN) signaling and functional responses by means of G-protein linked, 7-transmembrane domain (7-TMD) receptors. These systems always use cytosolic calcium ([Ca2+]i) fluxes as second messengers. We have previously shown that PMN sampled from trauma patients, as well as volunteer PMN, sequentially stimulated by chemotactic agents or exposed to fluids from sites of injury demonstrate altered calcium signaling. 1–4
“Nonexcitable” cells, such as immunocytes, endothelial cells, or hepatocytes do not express high-flux “voltage-operated” calcium channels. Instead, net [Ca2+]i flux is composed of contributions from multiple smaller calcium currents. Classically, G-protein activation of phospholipase C (PLC) cleaves inositol 1,4,5-triphosphate (IP-3) from membrane lipids: IP-3 diffuses rapidly to the endoplasmic reticulum (ER) where it initiates Ca2+ store release. Some 7-TMD receptors (e.g., for platelet activating factor, or PAF) are also thought to initiate receptor-mediated calcium entry through “receptor-operated” or “second messenger-operated” channels. 5 These events are very rapid, beginning within milliseconds of receptor ligation and typically peaking within 10 to 20 seconds. However, more prolonged [Ca2+]i signals occur when depletion of ER calcium stores gives rise to unknown signals which open membrane “store-operated” calcium influx (SOCI) channels. SOCI is the dominant calcium mobilizing event in myeloid cells: it begins after the peak transient and dissipates over several minutes. 6–8 All calcium influx mechanisms are dynamically opposed by sodium-calcium exchange (NCX) and calcium ATPases that either extrude calcium into the extracellular space or cause reuptake into ER stores. Interestingly, although NCX is normally a mechanism for calcium efflux, it has been reported to cause calcium influx under some conditions. 9
[Ca2+]i transients impact processes like motility, degranulation, and oxidative burst directly, but calcium also activates calmodulin and calcium-calmodulin–dependent kinases (CaM-K). These mediate a wider variety of cell responses. Thus, [Ca2+]i flux can act as an upstream activator of MAP kinases, NFκβ, and other transcription factors, respiratory burst and other crucial PMN systems. 10–12 The extent to which such actions depend on such specific characteristics of cell [Ca2+]i flux as peak [Ca2+]i, the duration of [Ca2+]i transients, or the mean cellular calcium activity (the area under the calcium concentration curve, or AUC) is unclear. However, it is now known that specific CaM-K isoforms can differentially transduce such “analog” [Ca2+]i transient events. 13 Thus, the prolongation of elevated cell calcium levels by SOCI is now well accepted as being of critical importance in many biologic systems, including the maintenance of vascular tone, glandular secretion, and PMN function (reviewed in Parekh and Penner). 6
In this study, we evaluated the morphology of [Ca2+]i signals generated by a variety of PMN agonists thought important in host responses to injury. Noting that more potent agonists were associated with prolonged increases in [Ca2+]i during the efflux phase of calcium signaling, we tested the hypothesis that PMN [Ca2+]i responses after injury might be enhanced by the depletion of cell Ca2+ stores and increased SOCI.
From the Department of Surgery, Division of Trauma, UMD-New Jersey Medical School, Newark, New Jersey.
Address for reprints: Carl J. Hauser MD, UMD/New Jersey Medical School, Department of Surgery, MSB G-524, 185 South Orange Avenue, Newark, NJ 07103; email: hausercj@UMDNJ.edu.
Submitted for publication September 24, 1999.
Accepted for publication December 31, 1999.
Supported in part by grants from the Foundation of UMD/New Jersey Medical School, and from the AO/ASIF Research Foundation, Basel, Switzerland.
Presented at the 59th Annual Meeting of the American Association for the Surgery of Trauma, September 16–18, 1999, Boston, Massachusetts.