Oral tungstate (Na2WO4) exposure reduces adaptive immune responses in mice after challenge.

Tungstate (WO²â»4) has been identified as a ground water contaminant at military firing ranges and can be absorbed by ingestion. In this study, C57BL6 mice were exposed to sodium tungstate (Na2WO4·2H2O) (0, 2, 62.5, 125, and 200 mg/kg/day) in their drinking water for an initial 28-day screen and in a one-generation (one-gen) model. Twenty-four hours prior to euthanasia, mice were intraperitoneally injected with Staphylococcal enterotoxin B (SEB) (20 µg/mouse) or saline as controls. After euthanasia, splenocytes and blood were collected and stained with lymphocyte and/or myeloid immunophenotyping panels and analyzed by flow cytometry. In the 28-day and one-gen exposure, statistically significant reductions were observed in the quantities of activated cytotoxic T-cells (TCTL; CD3(+)CD8(+)CD71(+)) and helper T-cells (TH; CD3(+)CD4(+)CD71(+)) from spleens of SEB-treated mice. In the 28-day exposures, CD71(+) TCTL cells were 12.87 ± 2.05% (SE) in the 0 tungstate (control) group compared to 4.44 ± 1.42% in the 200 mg/kg/day (p < 0.001) group. TH cells were 4.85 ± 1.23% in controls and 2.76 ± 0.51% in the 200 mg/kg/day (p < 0.003) group. In the one-gen exposures, TCTL cells were 7.98 ± 0.49% and 6.33 ± 0.49% for P and F1 mice after 0 mg/kg/day tungstate vs 1.58 ± 0.23% and 2.52 ± 0.25% after 200 mg/kg/day of tungstate (p < 0.001). Similarly, TH cells were reduced to 6.21 ± 0.39% and 7.20 ± 0.76%, respectively, for the 0 mg/kg/day P and F1 mice, and 2.28 ± 0.41% and 2.85 ± 0.53%, respectively, for the 200 mg/kg/day tungstate P and F1 groups (p < 0.001). In delayed-type hypersensitivity Type IV experiments, tungstate exposure prior to primary and secondary antigen challenge significantly reduced footpad swelling at 20 and 200 mg/kg/day. These data indicate that exposure to tungstate can result in immune suppression that may, in turn, reduce host defense against pathogens.

Early infection during burn-induced inflammatory response results in increased mortality and p38-mediated neutrophil dysfunction.

Following burn injury, the host is susceptible to bacterial infections normally cleared by healthy patients. We hypothesized that during the systemic immune response that follows scald injury, the host's altered immune status increases infection susceptibility. Using a murine model of scald injury under inhaled anesthesia followed by intraperitoneal infection, we observed increased neutrophil numbers and function at postburn day (PBD) 1 compared with sham-burned and PBD4 mice. Further, increased mortality, bacteremia, and serum IL-6 were observed in PBD1 mice after Pseudomonas aeruginosa (PA) infection compared with sham-burned and PBD4 mice infected with PA. To examine these disparate responses, we investigated neutrophils isolated at 5 and 24 h following PA infection from PBD1 and sham-burned mice. Five hours after infection, there was no significant difference in number of recruited neutrophils; however, neutrophils from injured mice had decreased activation, active-p38, and oxidative burst compared with sham-burned mice. In direct contrast, 24 h after infection, we observed increased numbers, active-p38, and oxidative burst of neutrophils from PBD1 mice. Finally, we demonstrated that in neutrophils isolated from PBD1 mice, the observed increase in oxidative burst was p38 dependent. Altogether, neutrophil activation and function from thermally injured mice are initially delayed and later exacerbated by a p38-dependent mechanism. This mechanism is likely key to the observed increase in bacterial load and mortality of PBD1 mice infected with PA.

Sodium tungstate (Na2WO4) exposure increases apoptosis in human peripheral blood lymphocytes.

The potential for adverse health effects of using tungsten and its alloys in military munitions are an important concern to both civilians and the US military. The toxicological implications of exposure to tungsten, its alloys, and the soluble tungstate (Na(2)WO(4)) are currently under investigation. To examine tungstate toxicity, a series of experiments to determine its in vitro effects on cells of the immune system were performed. We identified alterations in isolated human peripheral blood lymphocytes (PBL) treated in vitro with sodium tungstate (0.01, 0.1, 1.0, and 10 mM). Analyses of apoptosis with annexin V and propidium iodide revealed a dose- and time-dependent increase in the quantity of cells in early apoptosis after tungstate exposure. Reductions in the number of cells entering into the cell cycle were also noted. Exposure of PBL to tungstate (1 mM) and Concanavalin A (ConA) for 72 h reduced the number of cells in S and G(2)/M phases of the cell cycle. There were alterations in the numbers of cells in G(0)/G(1), S, and G(2)/M phases of the cell cycle in long-term THP-1 (acute leukemic monocytes) cultures treated with tungstate (0.01, 0.1, 1.0, and 10 mM). Gel electrophoresis, silver staining, and LC-MS/MS showed the cytoplasmic presence of histone H1b and H1d after 72 h of tungstate exposure. The addition of tungstate to cultures resulted in significant reductions in the quantity of interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha), and IL-6 produced by stimulated [CD3/CD28, ConA, or lipopolysaccharide (LPS)] and tungstate-treated lymphocytes. Taken together, these data indicate that tungstate increases apoptosis of PBL, alters cell cycle progression, reduces cytokine production, and therefore warrants further investigation.

Divergent adaptive and innate immunological responses are observed in humans following blunt trauma.

BACKGROUND: The immune response to trauma has traditionally been modeled to consist of the systemic inflammatory response syndrome (SIRS) followed by the compensatory anti-inflammatory response syndrome (CARS). We investigated these responses in a homogenous cohort of male, severe blunt trauma patients admitted to a University Hospital surgical intensive care unit (SICU). After obtaining consent, peripheral blood was drawn up to 96 hours following injury. The enumeration and functionality of both myeloid and lymphocyte cell populations were determined. RESULTS: Neutrophil numbers were observed to be elevated in trauma patients as compared to healthy controls. Further, neutrophils isolated from trauma patients had increased raft formation and phospho-Akt. Consistent with this, the neutrophils had increased oxidative burst compared to healthy controls. In direct contrast, blood from trauma patients contained decreased naïve T cell numbers. Upon activation with a T cell specific mitogen, trauma patient T cells produced less IFN-gamma as compared to those from healthy controls. Consistent with these results, upon activation, trauma patient T cells were observed to have decreased T cell receptor mediated signaling. CONCLUSIONS: These results suggest that following trauma, there are concurrent and divergent immunological responses. These consist of a hyper-inflammatory response by the innate arm of the immune system concurrent with a hypo-inflammatory response by the adaptive arm.

Alterations in membrane cholesterol cause mobilization of lipid rafts from specific granules and prime human neutrophils for enhanced adherence-dependent oxidant production.

The present study sought to examine the function of membrane lipid rafts in adherence-dependent oxidant production in human neutrophils. Rafts are membrane domains that are rich in glycosphingolipids and cholesterol and are thought to be the foci for formation of signaling complexes in a variety of cells. Disruption of lipid rafts by depletion of membrane cholesterol with the chelating agent methyl-beta-cyclodextrin (MbetaCD) has been widely used to examine the function of lipid rafts. Here, we report that treatment of human neutrophils with MbetaCD unexpectedly caused priming of these cells, manifested as enhanced adherence-dependent oxidant production. Treatment of neutrophils with MbetaCD dose-dependently increased oxidant production after adhesion to fibronectin-coated plates. This priming effect was associated with recruitment of CD11b- and CD66b-rich raft domains from the specific granules, as determined by immunoblot and flow cytometry. Confocal microscopy showed that MbetaCD caused otherwise untreated neutrophils to rapidly adhere and spread on fibronectin-coated plates. Furthermore, three-dimensional reconstruction microscopy studies showed that MbetaCD caused expansion and coalescence of raft domains that covered most of the cell surface. These large raft domains expressed CD11b primarily in the core of these regions. Our studies demonstrate that cholesterol depletion with MbetaCD results in neutrophil priming manifested as enhanced adherence-dependent oxidant production. These studies caution against assumption that any observed MbetaCD effects are a function of reduced raft formation.

Formation of focal adhesion-like structures in circulating human neutrophils after severe injury: triggering of a tissue-phase response in the vascular space.

Neutrophils play a key role in injury to the lung, kidney, liver, and gastrointestinal tract, often seen after major trauma. We evaluated the role of integrin-linked focal adhesions in the primed state, previously identified in peripheral blood neutrophils from severely injured patients. Immunoblot analysis of Triton-insoluble cell fractions revealed that total paxillin content was unchanged in comparison with that found in neutrophils from healthy volunteers, but phosphorylation of paxillin on tyrosine residue 118 was increased by more than 2-fold. Immunoprecipitation with antipaxillin and immunoblotting for proline-rich tyrosine kinase 2 (Pyk2) and for fgr showed significantly more colocalization. Densitometric analysis of total phosphotyrosine profiles also demonstrated significantly more in patient cells as compared with healthy cells. When allowed to adhere to fibronectin-coated plates, healthy and patient cells demonstrate a significant increase in tyrosine phosphorylation from that found in suspension-phase cells. Differential interference contrast microscopy of healthy neutrophils adherent to fibronectin matrices demonstrated rounded cells, without evidence of spreading; spreading was induced by addition of TNF-alpha. Patient neutrophils spread spontaneously, a response not further enhanced by TNF-alpha. Confocal imaging using anti-Pyk2 demonstrated aggregation of Pyk2 into punctate structures in patient but not in healthy cells. We conclude that neutrophils from severely injured patients are in a primed state, characterized by formation of focal adhesion-like structures. The identification of such structures in a clinical disease setting where they likely participate in unwanted consequences provides a novel area for study of regulation of neutrophil function.