CD8⁺ T-cell-mediated acute lung injury depends in part on CXCR2 signaling.

<p>WT and CXCR2^−/− SPC-HA transgenic mice received 5×10⁶ WT HA-specific CD8⁺ T cells via tail vein injection. (A) Survival of mice after T-cell transfer was monitored daily and a striking difference in survival (<i>P</i><0.001) was observed. Representative H&E stained lung sections from (B) WT-HA or (C) CXCR2^−/−-HA mice harvested 5 days after transfer of WT HA-specific CD8⁺ T cells shown at 10x magnification with 40x inset. Data are representative of at least two independent experiments with 4-5 mice per group.</p

CD8⁺ T-cell expression of ADAM17 is required for TNF-α processing following antigen recognition <i>in vivo</i>.

<p>SPC-HA transgenic mice received 10⁷ WT or ADAM17^−/− HA-specific CD8⁺ T cells via tail vein injection. ELISA was used to assess (A) TNF-α and (B) IFN-γ expression in cell-free BAL fluid. Alternatively, HA-specific CD8⁺ T cells were labeled with CFSE prior to transfer. Twenty-four hours after transfer, cells were recovered from (C) BAL and (D) lung homogenates and assessed by flow cytometric analysis to determine the total number of transferred HA-specific CD8⁺ T cells present. Data represent mean ± standard deviation. Data are representative of two or more independent experiments with 4 mice per group. **<i>P</i><0.01.</p

Processing of TNF-α by CD8⁺ T cells is required for severe and lethal lung injury.

<p>SPC-HA transgenic mice received WT or tmTNF HA-specific CD8⁺ T cells via tail vein injection. (A) Survival of mice after transfer of 10⁷ T cells was monitored and a significant difference in survival (<i>P</i><0.05) was observed. Representative H&E stained lung sections from SPC-HA transgenic mice harvested 5 days after transfer of 5×10⁶ (B) WT or (C) tmTNF CD8⁺ T cells shown at 10x magnification with 40x inset. (D) Carbon monoxide uptake was measured to assess lung function. (E) Percentage of neutrophils in whole lung homogenates was determined by morphological analysis of cells after staining cytospin preparations. (F) ELISA was used to assay CXCL2 expression in cell-free whole lung homogenates. Data represent mean ± standard deviation. Data are representative of at least two independent experiments with 3-4 mice per group. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

ADAM17 expression on transferred CD8⁺ T cells is required for enhanced airway inflammation.

<p>SPC-HA transgenic mice received 10⁷ WT or ADAM17^−/− HA-specific CD8⁺ T cells via tail vein injection. Twenty-four hours after T-cell transfer, cell-free BAL fluid was prepared and the levels of (A) CXCL2, (B) CCL2, (C) IP-10, (D) CCL11, (E) CXCL5, and (F) G-CSF were determined by Luminex assay. Data represent mean ± standard deviation. Data are representative of two mice from two independent experiments with a total of 4 mice per group. *<i>P</i><0.05, **<i>P</i><0.01.</p

CD8⁺ T-cell processing of TNF-α is required for lung epithelial cell expression of CXCL2.

<p>MLE-K^d cells were pulsed with HA_210−219 peptide and co-cultured with HA-specific CD8⁺ T cells for 5 hours. In some experiments, recombinant mouse TNF-α was added to the culture supernatant. Cell-free supernatant was analyzed for expression of (A) TNF-α, (C) CCL2, and (D) CXCL2 by ELISA. (B) Alternatively, total TNF-α production by HA-specific CD8⁺ T cells after PMA/ionomycin stimulation with protease inhibitor and detergent solubilization was measured by ELISA. Data represents mean ± standard deviation. Data are representative of three independent experiments with each condition conducted in triplicate. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

ADAM17 expression on transferred CD8⁺ T cells is required for acute lung injury.

<p>SPC-HA transgenic mice received WT or ADAM17^−/− HA-specific CD8⁺ T cells via tail vein injection. (A) Survival of mice after transfer of 10⁷ T cells was monitored and a difference in survival (<i>P</i><0.05) was observed. Representative H&E stained lung sections from SPC-HA transgenic mice harvested 5 days after transfer of 5×10⁶ (B) WT or (C) ADAM17^−/− CD8⁺ T cells shown at 10x magnification with 40x inset. (D) ELISA was used to assess the levels of albumin in cell-free BAL fluid after transfer of 10⁷ T cells. (E) Peripheral oxygen saturation was measured in mice before and after transfer of 10⁷ T cells using the MouseOx System. Data represent mean ± standard deviation. Data are representative of at least three independent experiments with 3-4 mice per group. *<i>P</i><0.05, **<i>P</i><0.01.</p

Exposure to Arsenic during gestation and lactation affect total TG levels in the serum and liver.

<p>Asterisks indicate statistical significance,</p>*<p>p<0.05 and</p>**<p>p<0.01, compared to respective control. Values represent mean ± SEM. Two tailed student's t-test for gestational exposure; One Way ANOVA for post natal exposures.</p

Effects of arsenic exposure on the distribution of cholesterol and triglycerides over serum lipoprotein fractions.

<p>Dams were fasted for 6 hours on PN day 21 and pooled serum of 4 As (IU&PN) treated dams (open triangles) and 3 controls (black circles) was used for lipoprotein profiling by FPLC. Fractions were assayed for TG (top panel) and total cholesterol levels (bottom panel).</p

Arsenic measurements^a in tissue samples from dams and pups.

a<p>Total As levels were measured by ICP-MS, as described in Methods (n = 3–6). Values represent mean ± SEM. Asterisks indicate statistical significance from respective control group in matching row.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001. Number sign indicates statistical significance from virgin female mouse exposed to 10 ppb As for 2 weeks. One Way ANOVA.</p

Experimental model of developmental arsenic exposure in C57BL/6 mice.

<p>Following the detection of cervical plugs, mated females were exposed to control water or 10 ppb As in drinking water through the gestational period. At the birth of their pups, dams in each exposure group were further divided into sub-groups receiving control water or 10 ppb As in drinking water through 30 days of age (n = 14–17 dams per exposure). Weaning from the dam took place at day 21 PN (or later if a pup did not reach the 7 gram weight cut-off). This resulted in 4 overall exposure groups: 1. Control (no As drinking water exposure; 2. Postnatal (PN, offspring receiving 10 ppb As from PN days 1–30); 3. In utero (IU, offspring receiving 10 ppb As from gestational day 1 through birth); 4. In utero & postnatal (IU&PN, offspring receiving 10 ppb As from gestational day 1 through day 30 PN) At day 30 PN, all offspring were placed on control drinking water and growth was assessed until 6 weeks PN.</p