Thyroid nodules in xeroderma pigmentosum patients: a feature of premature aging.

PURPOSE: Xeroderma pigmentosum (XP) is an autosomal recessive disease with defective DNA repair, a markedly increased risk of skin cancer, and premature aging. Reports from North Africa have described thyroid nodules in XP patients, but thyroid nodule prevalence has never been determined in XP patients enrolled in our natural history study at the National Institutes of Health (NIH). METHODS: We performed thyroid ultrasound examinations on all 29 XP patients examined from 2011 to 2019 and assessed nodule malignancy using the Thyroid Imaging Reporting and Data System. Thyroid nodule prevalence was also obtained from comparison cohorts. DNA sequencing was performed on thyroid tissue from XP patients who had surgery for thyroid cancer. RESULTS: Thyroid nodules were identified in 18/29 XP patients (62%). The median age of patients with thyroid nodules in our XP cohort (20 years) was younger than that of three comparison groups: 36 years (California study-208 subjects), 48 years (Korean study-24,757 subjects), and 52 years (NIH-682 research subjects). Multiple (2-4) thyroid nodules were found in 12/18 (67%) of the patients with nodules. Autopsy examination revealed follicular adenomas in 4/8 (50%) additional XP patients. DNA sequencing revealed rare mutations in two other XP patients with papillary thyroid cancer. CONCLUSIONS: XP patients have an increased incidence of thyroid nodules at an early age in comparison to the general population. These finding confirm another premature aging feature of XP.

Activin A inhibits vascular endothelial cell growth and suppresses tumour angiogenesis in gastric cancer.

BACKGROUND: Activin A is a multi-functional cytokine belonging to the transforming growth factor-ß (TGF-ß) superfamily; however, the effect of activin A on angiogenesis remains largely unclear. We found that inhibin ß A subunit (INHBA) mRNA is overexpressed in gastric cancer (GC) specimens and investigated the effect of activin A, a homodimer of INHBA, on angiogenesis in GC. METHODS: Anti-angiogenic effects of activin A via p21 induction were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro and a stable INHBA-introduced GC cell line in vivo. RESULTS: Compared with TGF-ß, activin A potently inhibited the cellular proliferation and tube formation of HUVECs with induction of p21. A promoter assay and a chromatin immunoprecipitation assay revealed that activin A directly regulates p21 transcriptional activity through Smads. Stable p21-knockdown significantly enhanced the cellular proliferation of HUVECs. Notably, stable p21-knockdown exhibited a resistance to activin-mediated growth inhibition in HUVECs, indicating that p21 induction has a key role on activin A-mediated growth inhibition in vascular endothelial cells. Finally, a stable INHBA-introduced GC cell line exhibited a decrease in tumour growth and angiogenesis in vivo. CONCLUSION: Our findings highlight the suppressive role of activin A, unlike TGF-ß, on tumour growth and angiogenesis in GC.

Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development.

The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10(-6)) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offspring (n = 24) with respect to abnormalities during their antenatal and neonatal periods to pregnancies resulting in their unaffected siblings (n = 18), accounting for correlation, and to population reference values. Significantly higher incidence of several severe gestational complications was noted in TTD-affected pregnancies. Small for gestational age (SGA) <10th percentile [Relative risk (RR ) = 9.3, 95% CI = 1.4-60.5, p = 0.02], SGA <3rd percentile (RR = 7.2, 95% CI = 1.1-48.1, p = 0.04), and neonatal intensive care unit (NICU) hospitalization (RR = 6.4, 95% CI = 1.4-29.5, p = 0.02) occurred more frequently among TTD-affected neonates compared with their unaffected siblings. Compared with reference values from general obstetrical population, pregnancies that resulted in TTD-affected infants were significantly more likely to be complicated by hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (RR = 35.7, 95% CI = 7.6-92.5, p = 0.0002), elevated mid-trimester maternal serum human chorionic gonadotropin (hCG) levels (RR = 14.3, 95% CI = 7.0-16.6, p < 0.0001), SGA <3rd percentile (RR = 13.9, 95% CI = 7.4-21.1, p < 0.0001), pre-term delivery (<32 weeks) (RR = 12.0, 95% CI = 4.9-21.6, p < 0.0001), pre-eclampsia (RR = 4.0, 95% CI = 1.6-7.4, p = 0.006), and decreased fetal movement (RR = 3.3, 95% CI = 1.6-5.2, p = 0.0018). Abnormal placental development is an underlying mechanism that may explain the constellation of observed complications in our study. Thus, we hypothesize that TTD DNA repair and transcription genes play an important role in normal human placental development.

Trichothiodystrophy: a systematic review of 112 published cases characterises a wide spectrum of clinical manifestations.

Trichothiodystrophy (TTD) is a rare, autosomal recessive disease, characterised by brittle, sulfur deficient hair and multisystem abnormalities. A systematic literature review identified 112 patients ranging from 12 weeks to 47 years of age (median 6 years). In addition to hair abnormalities, common features reported were developmental delay/intellectual impairment (86%), short stature (73%), ichthyosis (65%), abnormal characteristics at birth (55%), ocular abnormalities (51%), infections (46%), photosensitivity (42%), maternal pregnancy complications (28%) and defective DNA repair (37%). There was high mortality, with 19 deaths under the age of 10 years (13 infection related), which is 20-fold higher compared to the US population. The spectrum of clinical features varied from mild disease with only hair involvement to severe disease with profound developmental defects, recurrent infections and a high mortality at a young age. Abnormal characteristics at birth and pregnancy complications, unrecognised but common features of TTD, suggest a role for DNA repair genes in normal fetal development.

Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: a complex genotype-phenotype relationship.

Patients with the rare genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) have defects in DNA nucleotide excision repair (NER). The NER pathway involves at least 28 genes. Three NER genes are also part of the basal transcription factor, TFIIH. Mutations in 11 NER genes have been associated with clinical diseases with at least eight overlapping phenotypes. The clinical features of these patients have some similarities but also have marked differences. NER is involved in protection against sunlight-induced DNA damage. While XP patients have 1000-fold increase in susceptibility to skin cancer, TTD and CS patients have normal skin cancer risk. Several of the genes involved in NER also affect somatic growth and development. Some patients have short stature and immature sexual development. TTD patients have sulfur deficient brittle hair. Progressive sensorineural deafness is an early feature of XP and CS. Many of these clinical diseases are associated with developmental delay and progressive neurological degeneration. The main neuropathology of XP is a primary neuronal degeneration. In contrast, CS and TTD patients have reduced myelination of the brain. These complex neurological abnormalities are not related to sunlight exposure but may be caused by developmental defects as well as faulty repair of DNA damage to neuronal cells induced by oxidative metabolism or other endogenous processes.

Resuscitation with a blood substitute abrogates pathologic postinjury neutrophil cytotoxic function.

BACKGROUND: Resuscitation with oxygen-carrying fluids is critically important in the patient with hemorrhagic shock caused by trauma. However, it is clear that a number of biologic mediators present in stored blood (packed red blood cells [PRBCs]) have the potential to exacerbate early postinjury hyperinflammation and multiple organ failure through priming of circulating neutrophils (PMNs). PolyHeme (Northfield Laboratories, Evanston, IL), a hemoglobin-based substitute that is free of priming agents, provides an alternative. We hypothesized that PMN priming would be attenuated in patients resuscitated with PolyHeme in lieu of stored blood. METHODS: Injured patients requiring urgent transfusion were given either PolyHeme (up to 20 units) or PRBCs. Early postinjury PMN priming was measured via beta-2 integrin expression, superoxide production, and elastase release. RESULTS: Treatment groups were comparable with respect to extent of injury and early physiologic compromise. PMNs from patients resuscitated with PRBCs showed priming in the early postinjury period by all three measures. No such priming was evident in patients resuscitated with PolyHeme. CONCLUSION: The use of a blood substitute in the early postinjury period avoids PMN priming and may thereby provide an avenue to decrease the incidence or severity of postinjury multiple organ failure.

Insulin resistance in patients with depression and its changes during the clinical course of depression: minimal model analysis.

A high proportion of patients with depression develop glucose intolerance accompanied by hyperinsulinemia, suggestive of reduced insulin sensitivity (insulin resistance). The aim of this study was to evaluate insulin sensitivity in patients with depression and its changes during the clinical course of depression. Twenty nondiabetic patients with depression (13 males and 7 females aged 44+/-14 years; body mass index [BMI] 23.2+/-2.8 kg/m2) were prospectively studied by the frequently sampled intravenous glucose tolerance test (FSIGT) and the oral glucose tolerance test (OGTT) before and after treatment of depression, and an age-, sex-, and BMI-matched control group (n = 13) was examined once by the FSIGT. Metabolic indices measuring glucose effectiveness at basal insulin (SG) and insulin sensitivity (SI) were derived from minimal model analysis. Each patient was treated by cyclic antidepressants with an 1,800 to 2,200 kcal/d food intake and underwent no exercise therapy. SI was significantly lower in patients before treatment versus control subjects (6.0+/-2.5 v 13.8+/-8.6 x 10(-5) min(-1) x mol(-1) x L, P < .01). After treatment of depression, a significant increase in SI (10.7+/-7.5 x 10(-5) min(-1) x mol(-1) x 1, P

Maximal human neutrophil priming for superoxide production and elastase release requires p38 mitogen-activated protein kinase activation.

HYPOTHESIS: Neutrophil priming has been implicated in the development of multiple organ failure, although the precise intracellular mechanisms that regulate neutrophil priming remain unclear. Our previous work characterized platelet-activating factor (PAF) priming of human neutrophils for concordant superoxide anion (O2-) generation and elastase degranulation. The p38 mitogen-activated protein kinase (MAPK) is activated by PAF stimulation. We hypothesized that PAF-induced human neutrophil priming for O2- and elastase release is mediated via the p38 MAPK pathway. DESIGN: Isolated neutrophils from 6 human donors were preincubated with the specific p38 MAPK inhibitor SB 203580 (1 micromol/L) or buffer (control) for 30 minutes. Cells were then primed with PAF (200 nmol/L), followed by receptor-dependent (N-formyl-methionyl-leucyl-phenylalanine, 1 micromol/L) or receptor-independent phorbol myristate acetate (PMA, 100 ng/mL) activation. SETTING: Urban trauma research laboratory. PATIENTS: Healthy volunteer donors of neutrophils. MAIN OUTCOME MEASURES: Maximal rate of O2- generation was measured by superoxide dismutase-inhibitable reduction of cytochrome c and elastase release by the cleavage of N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. RESULTS: SB 203580 significantly attenuated the generation of O2- and release of elastase from neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine but not with PMA. Independent of the activator receptor status, SB 203580 almost completely blocked the exaggerated neutrophil cytotoxic response due to PAF priming. CONCLUSIONS: The p38 MAPK pathway is required for maximal PAF-induced neutrophil priming for O2- production and elastase degranulation. Therefore, the MAPK signaling cascade may offer a potential therapeutic strategy to preempt global neutrophil hyperactivity rather than attempt to nullify the end products independently.

Hypertonic saline resuscitation abrogates neutrophil priming by mesenteric lymph.

OBJECTIVE: Neutrophil (PMN) priming after hemorrhagic shock is predictive of the subsequent development of multiple organ failure, but the mechanism remains unknown. Recently, we and others have demonstrated that mesenteric lymph from shock animals resuscitated with lactated Ringer's solution (LR) is not only a potent PMN priming agent but also causes lung injury. Work by others has shown that resuscitation with hypertonic saline (HTS) protects animals from lung injury after hemorrhagic shock. Therefore, we hypothesize that resuscitation with HTS will abolish PMN priming by postshock mesenteric lymph. METHODS: After mesenteric lymph duct catheterization, male rats underwent hemorrhagic shock (mean arterial pressure of 40 mm Hg for 90 minutes) and resuscitation with shed blood plus either LR (2x volume of shed blood) or 4 mL/kg of 7% HTS (isonatremic). Priming for superoxide by PMN was measured after fMLP (1 microM) activation. RESULTS: Shock significantly decreased mesenteric lymph flow from preshock levels in both groups. LR resuscitation produced significantly more mesenteric lymph than HTS resuscitation. Mesenteric lymph from LR animals primed PMN for superoxide production, whereas, HTS eliminated this priming. CONCLUSION: HTS not only decreases postshock mesenteric lymph production, it eliminates PMN priming by mesenteric lymph, suggesting a mechanism for the beneficial effects of HTS resuscitation.

Extracellular signal-related kinase 1/2 and p38 mitogen-activated protein kinase pathways serve opposite roles in neutrophil cytotoxicity.

BACKGROUND: Inflammatory stimuli rapidly activate mitogen-activated protein kinases (MAPKs) in neutrophils (PMNs). However, their role in cytotoxic function remains unknown. Elucidating the signals involved in release of cytotoxic agents from PMNs may provide new avenues for therapy in diseases of diminished or excessive PMN function. HYPOTHESIS: The p38 MAPK and extracellular signal-related kinase 1/2 (ERK1/2) modulate superoxide generation and elastase release in activated human PMNs. STUDY DESIGN: Isolated human PMNs were incubated with specific inhibitors of MAPK pathways, or vehicle control solution, before activation with the bacterial peptide f-Met-Leu-Phe. MAIN OUTCOME MEASURES: The rate of superoxide release from activated PMNs was measured by the superoxide dismutase-inhibitable reduction of cytochrome-c. Elastase release from PMNs was determined by cleavage of the substrate Ala-Ala-Pro-Val-pNA. RESULTS: Superoxide release from activated PMNs was inhibited by blockade of p38 MAPK activation but unaffected by blockade of ERK1/2. Conversely, elastase release was unaffected by p38 MAPK inhibition and increased by ERK1/2 inhibition. CONCLUSIONS: Activation of p38 MAPK promotes superoxide release from PMNs activated by f-Met-Leu-Phe. The ERK1/2 pathway may serve as a negative feedback mechanism for granule exocytosis.

Posthemorrhagic shock mesenteric lymph primes circulating neutrophils and provokes lung injury.

Mesenteric lymph has recently been invoked as an avenue for gut-derived factors that may result in distant organ injury following hemorrhagic shock. We demonstrate that posthemorrhagic shock mesenteric lymph primes neutrophils (PMNs) and causes lung injury. Methods. Mesenteric lymph was collected from Sprague-Dawley rats from their mesenteric lymph duct prior to, during, and following hemorrhagic shock (MAP 40 for 90 min). The rats were then resuscitated with shed blood plus lactated Ringers (2X shed blood) over 3 h. Lung leak was assessed by transudation of Evan's blue dye into the alveolus as measured by bronchoalveolar lavage. Isolated human PMNs were incubated with 1 and 10% lymph; priming was measured by the fMLP (1 microM)-stimulated production of superoxide and surface expression of CD11b determined by flow cytometry. Results. Mesenteric lymph flow increased significantly during resuscitation: preshock 144.4 microl/h, shock 44.5 microl/h, resuscitation 566.6 microl/h. Furthermore, diversion of this lymph abrogated lung injury as compared to rats without lymph diversion. Finally, mesenteric lymph from postshock animals primed PMNs for superoxide production (nearly three times control cells) as well as increased surface expression of CD11b (2-fold over control). Conclusion. Mesenteric lymph primes PMNs and causes lung injury following hemorrhagic shock. Mesenteric lymph provides a conduit for proinflammatory mediators that may participate in the pathogenesis of MOF.

Disparities in the respiratory burst between human and rat neutrophils.

The importance of reactive oxygen species (ROS) in neutrophil (PMN)-mediated injury to host tissues has been strongly implicated in a number of animal models. Peculiarities of the laboratory rat PMN, including an apparent paucity of superoxide release, prompted us to examine disparities in the respiratory burst between human and rat PMNs. Using isolated PMNs, we examined oxygen consumption, superoxide release, nitrate/nitrite release, and dihydrorhodamine (DHR) oxidation in response to an array of soluble stimuli. Our findings confirm that intact rat PMNs release little superoxide in comparison to human PMNs when primed and activated by soluble stimuli. For example, PMA-activated human PMNs released superoxide at 10.1 +/- 2.7 times the rate of rat PMNs (P < 0.01). However, measurements of oxygen consumption, cell-associated oxidant production (by DHR oxidation) and release of superoxide from electroporated cells suggests that rat PMNs generate oxidants at rates equivalent to human PMNs but preferentially release them in an intracellular compartment. Implications for the study of PMN-mediated oxidant injury in animal models are discussed.

Circulating postinjury neutrophils are primed for the release of proinflammatory cytokines.

BACKGROUND: Postinjury neutrophil (PMN) priming identifies the injured patient at risk for the subsequent development of multiple organ failure (MOF). PMN priming has previously been shown to cause enhanced release of proteases and superoxide. PMNs, however, are a rich source of proinflammatory cytokines, such as interleukin (IL)-8 and tumor necrosis factor (TNF), which have been implicated in the development of MOF. PMNs also make IL-1ra, which is an anti-inflammatory cytokine that inhibits IL-1. It is our hypothesis that postinjury PMNs are primed for increased stimulated release of the proinflammatory cytokines IL-8 and TNF but not the anti-inflammatory cytokine IL-1ra. METHODS: Twelve trauma patients with a mean Injury Severity Score of 24 (+/-4.6) and 10 elective surgical patients were studied. Postinjury PMNs were isolated from blood obtained at presentation (within 2 hours after injury) and 24 hours after trauma. PMNs from elective surgical patients were obtained preoperatively, immediately postoperatively, and at 24 hours. The PMNs were stimulated with platelet-activating factor (200 nM)/N-formyl-methionyl-leucyl-phenylalanine (1 micromol/L) or lipopolysaccharide (100 ng/mL) incubated for 24 hours in RPMI-1640, and release of IL-8, TNF, and IL-1ra were measured. RESULTS: Postinjury PMNs were primed for both platelet-activating factor/N-formyl-methionyl-leucyl-phenylalanine-stimulated and lipopolysaccharide-stimulated IL-8 and TNF release at 2 hours after injury (fourfold increase of IL-8 release and fivefold increase of TNF release), whereas elective surgical patients demonstrated no priming. In contrast, postinjury patients were not primed for increased release of the counterinflammatory cytokine IL-1ra, suggesting a specific postinjury up-regulation of IL-8 and TNF. CONCLUSION: After injury, PMNs are primed for proinflammatory cytokine release in addition to superoxide and elastase. This augmented release of IL-8 and TNF may be involved in the subsequent development of organ dysfunction and ultimately MOF.

Postinjury suppression of human neutrophil cytokine production results from the stabilization of inhibitory kappaB.

UNLABELLED: Postinjury neutrophil (PMN) dysfunction is a well recognized event that may be responsible for increased infections. PMN cytokine production is an important component of their bactericidal capacity. When PMNs are stimulated, inhibitory factor kappaB (IkappaB) is degraded, allowing nuclear factor kappaB (NFkappaB) to translocate to the nucleus and promotes genes for the transcription of the interleukin-8 (IL-8) and tumor necrosis factor (TNF) genes. We hypothesize that similar to their late postinjury depressed superoxide production, postinjury PMNs manifest suppressed cytokine production, which is mediated by stabilization of IkappaB levels. METHODS: Twelve severely injured patients with an injury severity score (ISS) of 24 (+/-4.6) were studied as well as 10 elective surgical patients as a control. PMNs were isolated and incubated for 24 h in RPMI. PMNs were stimulated with lipopolysaccharide (LPS; 100 ng) or PAF (200 nm) and fMLP (1 microM) and release of IL-8, TNF, and interleukin-1 receptor antagonist (IL-1ra) were measured. Postinjury PMNs were also stimulated with LPS (100 ng), and IkappaB breakdown was measured at 0, 30, and 60 min using gel electrophoresis. RESULTS: Postinjury PMNs displayed a significant suppression of both IL-8 and TNF on postinjury Days 1-3, while the release of IL-1ra was preserved throughout the entire study period. In contrast, elective surgical patients demonstrated no decrease in IL-8 or TNF. Furthermore, IkappaB levels were preserved in the postinjury PMNs as compared with normal control PMNs. CONCLUSION: Postinjury PMNs have a suppressed release of both IL-8 and TNF following injury that did not occur in elective surgical patients. Furthermore, the NFkappaB/IkappaB-independent IL-1ra did not show suppression of release. In addition, stabilization of IkappaB following severe injury leads to decreased PMN IL-8 and TNF production. This genetic reprogramming may help explain PMN dysfunction and subsequent infections seen in severely injured patients.

New mechanisms by which secretory phospholipase A2 stimulates neutrophils to provoke the release of cytotoxic agents.

BACKGROUND: Secretory phospholipase A2 (sPLA2) is a potent proinflammatory enzyme that stimulates inflammation through the production of reactive lipids. However, enzymatic inhibitors have been disappointing in their effectiveness in halting hyperinflammation. OBJECTIVE: To determine whether sPLA2 acts directly on neutrophil plasma membrane lipids or via a nonenzymatic mechanism. DESIGN: Isolated neutrophils (PMNs) were incubated with 3 types of sPLA2, and elastase and superoxide release from PMNs was measured. Ethyleneglycotetraacetic acid was used as a selective enzymatic inhibitor. The PMNs were exposed to sPLA2 in the presence and absence of ethyleneglycotetraacetic acid and the release of elastase was measured. SETTING: Urban trauma research laboratory. PATIENTS: Normal healthy donors of PMNs. MAIN OUTCOME MEASURES: Stimulated release of superoxide and elastase. RESULTS: The sPLA2 acted directly on plasma membrane lipids to stimulate the PMN to produce superoxide and release elastase. This mechanism is blocked with enzymatic inhibition of sPLA2. The sPLA2 also provokes elastase release from PMNs independently of its enzymatic function. This mechanism is not blocked with traditional enzymatic inhibitors. CONCLUSIONS: These data indicate that the sPLA2 can act directly on PMNs to stimulate the release of inflammatory mediators via enzymatic degradation of plasma membrane lipids. In addition, sPLA2 can act as a ligand and stimulate the PMN independently of its enzymatic activity.

Interleukin-6 augments neutrophil cytotoxic potential via selective enhancement of elastase release.

BACKGROUND: Interleukin-6 (IL-6) appears to be a reliable marker of disease severity in critically ill patients at risk for inflammatory organ injury such as ARDS and MOF. Debate continues, however, as to whether this pleiotropic cytokine acts principally as a proinflammatory or counterregulatory mediator. Because the polymorphonuclear leukocyte (PMN) is a central effector of inflammatory injury, defining the effects of IL-6 on mechanisms of PMN cytotoxicity may be revealing. Previous investigations of PMN release of reactive oxygen species demonstrate that IL-6 in concert with other mediators may augment cytotoxicity. We hypothesized that IL-6 alone increases PMN cytotoxic potential through selective enhancement of elastase release. MATERIALS AND METHODS: Isolated human PMNs were incubated with IL-6 in the physiologic range observed in critically ill patients (0.1 to 100 ng/ml) for 10 to 30 min. Selected cells were then activated with fMLP (1 microM). Elastase release was measured by specific cleavage of AAPV-pNA and compared to untreated cells and cells activated with formyl-Met-Leu-Phe (fMLP; 1 microM) alone. To determine if changes in elastase release might be due to IL-6 induced generation of PAF, WEB 2347 (50 microM) was preincubated with selected cells for 20 min. Surface expression of beta 2 integrins was measured by flow cytometry after incubating with labeled antibodies to CD11b and CD18. RESULTS: IL-6 alone at 100 ng/ml augmented basal elastase release by 116 +/- 41% within 10 min. Doses as low as 0.1 ng/ml stimulated elastase release when the incubation time was increased to 30 min. After 30 min of incubation, IL-6 at all doses examined augmented the elastase release of fMLP-activated cells (increases of 33 to 45%). WEB 2347 preincubation did not block augmentation of elastase release by IL-6 at 10 ng/ml. IL-6 had no effect on surface expression of beta 2 integrins at 10 ng/ml. CONCLUSIONS: IL-6 alone enhances both basal and fMLP-stimulated elastase release by PMNs. This proinflammatory action on PMNs may help explain the observed correlation between circulating IL-6 levels and inflammatory organ injury.

Primed neutrophils require phosphatidic acid for maximal receptor-activated elastase release.

BACKGROUND: Priming of neutrophils (PMNs) for protease release is believed to be central to the pathogenesis of PMN-mediated tissue injury observed in ARDS/MOF. Defining the intracellular signaling pathways involved with this excessive protease release may aid in establishing future therapies for ARDS. Phospholipase D (PLD) production of phosphatidic acid (PA) is thought to be pivotal in reactive oxygen species generation but its role in degranulation (i. e., protease release) remains unclear. Our hypothesis was that primed neutrophils require PLD production of PA for maximal activated release of elastase. METHODS: Isolated human PMNs were incubated with a well described antagonist of PA production, ethanol (ETOH, 100-1000 mg/dL), and then primed (PAF, 200 nM) followed by activation (fMLP, 1 microM). To mimic fMLP receptor-dependent activation, PMNs were primed and then directly activated with exogenous dioctanoyl l-alpha-phosphatidic acid (PA8, 0.5-200 microM). To confirm the importance of PA in elastase release, PA8 was given to primed-activated PMNs after ethanol pretreatment in an attempt to recover the maximal response. Elastase release was measured by the cleavage of AAPV-pNA. RESULTS: PA blockade with ETOH attenuated PAF-primed/fMLP-activated PMN elastase release in a dose-dependent manner. Exogenous PA8 reproduced maximally primed-activated PMN elastase release, and furthermore, PA8 was able to restore maximal elastase release following ethanol attenuation. CONCLUSIONS: Elastase release from PAF-primed/ fMLP-activated neutrophils is dependent on PA production. Thus, PA production appears to be involved in both oxidant-dependent and independent mechanisms of neutrophil cytotoxicity and may be a potential therapeutic target in the treatment of hyperinflammatory diseases such as ARDS/MOF.