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Transgenic HbS Mouse Neutrophils in Increased Susceptibility to Acute Lung Injury
whereas IFN-g augmented p42mapk/erk2 and p46 JNK activation by TNF-a, IL-4 downregulated p42mapk/erk2 and p46 JNK activation by TNF-a. Treatment of cells with N-acetylcysteine (NAC), previously shown to inhibit the c-Jun N-terminal kinase/stressactivated protein kinase (JNK/SAPK) pathway between TNF receptor-associated factor-2 (TRAF2) and MAPK/ ERK kinase kinase (MEKK), revealed that both iNOS (Fig 1) and NO22 expression were significantly inhibited. However, NAC had no effect on IRF-1 messenger RNA expression stimulated by IFN-g and no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts (Fig 1). Furthermore, NAC prevented the characteristic shape change in the macrophages when stimulated with TNF-a and IFN-g. Because NAC also inhibited p38mapk and p42mapk/erk2 activation by TNF-a in addition to inhibiting p46 JNK activation, the cells were treated with a specific MEK1 inhibitor (PD98059) and/or a p38mapk inhibitor (SB203580), and these inhibitors were found to have no effect on NO22 production by TNF-a and IFN-g. Preliminary transfections with 3T3 fibroblasts with the iNOS promoter;luciferase reporter constructs and dominant-negative MEKK1 suggest that the DN-MEKK1 inhibits TNF-a 1 IFN-g induction of iNOS (Fig 2). Gel retardation assays in mouse MB revealed that NAC markedly inhibits NF-kB activation and modestly suppresses AP-1 activation by TNF-a. Combined with more recent studies suggesting that MEKK and/or JNK may enhance the activation of NF-kB, we propose that MEKK3 JNKK3 JNK signal transduction pathway is involved in the activation of iNOS by TNF-a and IFN-g by the activation of NF-kB. Speculatively, NF-kB may act synergistically with IRF-1 to enhance iNOS transcription by TNF-a and IFN-g.
Transgenic HbS Mouse Neutrophils in Increased Susceptibility to Acute Lung Injury* Implications for Sickle Acute Chest Syndrome L. Hsu, MD, PhD; T. McDermott; L. Brown; S. M. Aguayo, MD, FCCP
(CHEST 1999; 116:92S) he sickle cell acute chest syndrome (ACS) has many clinical similarities to other forms of acute lung injury such as ARDS. We investigated a role for neutrophils in ACS pathophysiology, using a transgenic (Tg) mouse expressing human sickle hemoglobin that has mild features of sickle cell disease under baseline conditions.
T
Hypothesis After experimental acute lung insult, this Tg HbS mouse will have increased pulmonary neutrophils compared with normal mice given the same acute insult.
Materials and Methods Mice received either oleic acid (OA) injection or sham injection of the tail vein, and 4 h later were exsanguinated with anesthetic overdose. To measure myeloperoxidase (MPO), as an indicator of the granulocytes present within the lungs, whole lungs were homogenized and the supernatant was assayed spectrophotometrically. For histochemical staining, lungs were fixed with formalin-acetone and embedded in paraffin.
Results The Tg HbS mice with OA injection (n 5 7) had significantly higher MPO than normal mice with OA injection (n 5 6) (0.038 compared with 0.018, p 5 0.02). OA-injected mouse lungs have neutrophils infiltrating lung interstitium as well as neutrophils still within alveolar capillaries. Lung MPO did not correlate with granulocyte count, and total WBC counts were similar for Tg HbS and normal mice after OA.
Conclusion Tg HbS mice have increased neutrophils after acute lung injury, compared to normal mice with the same OA insult. Despite the similar baseline conditions of lungs and neutrophils in Tg HbS mice, the presence of HbS is associated with susceptibility to acute lung injury. These animal model results may be a first step toward understanding the interaction between RBCs, endothelium, and WBCs in sickle-cell ACS.
Figure 2. DN-MEKK1 inhibits TNF-a 1 IFN-g induction of iNOS.
*From Atlanta Veterans Administration Medical Center and Emory University, Atlanta, GA. Correspondence to: Lewis Hsu, MD, PhD, Emory University School of Medicine, 69 Butler St SE, Atlanta, GA 30303
92S
Thomas L. Petty 41st Annual Aspen Lung Conference: Acute Lung Injury
Transgenic HbS Mouse Neutrophils in Increased Susceptibility to Acute Lung Injury* Implications for Sickle Acute Chest Syndrome L. Hsu, MD, PhD; T. McDermott; L. Brown; S. M. Aguayo, MD, FCCP
(CHEST 1999; 116:92S) he sickle cell acute chest syndrome (ACS) has many clinical similarities to other forms of acute lung injury such as ARDS. We investigated a role for neutrophils in ACS pathophysiology, using a transgenic (Tg) mouse expressing human sickle hemoglobin that has mild features of sickle cell disease under baseline conditions.
T
Hypothesis After experimental acute lung insult, this Tg HbS mouse will have increased pulmonary neutrophils compared with normal mice given the same acute insult.
Materials and Methods Mice received either oleic acid (OA) injection or sham injection of the tail vein, and 4 h later were exsanguinated with anesthetic overdose. To measure myeloperoxidase (MPO), as an indicator of the granulocytes present within the lungs, whole lungs were homogenized and the supernatant was assayed spectrophotometrically. For histochemical staining, lungs were fixed with formalin-acetone and embedded in paraffin.
Results The Tg HbS mice with OA injection (n 5 7) had significantly higher MPO than normal mice with OA injection (n 5 6) (0.038 compared with 0.018, p 5 0.02). OA-injected mouse lungs have neutrophils infiltrating lung interstitium as well as neutrophils still within alveolar capillaries. Lung MPO did not correlate with granulocyte count, and total WBC counts were similar for Tg HbS and normal mice after OA.
Conclusion Tg HbS mice have increased neutrophils after acute lung injury, compared to normal mice with the same OA insult. Despite the similar baseline conditions of lungs and neutrophils in Tg HbS mice, the presence of HbS is associated with susceptibility to acute lung injury. These animal model results may be a first step toward understanding the interaction between RBCs, endothelium, and WBCs in sickle-cell ACS.
Figure 2. DN-MEKK1 inhibits TNF-a 1 IFN-g induction of iNOS.
*From Atlanta Veterans Administration Medical Center and Emory University, Atlanta, GA. Correspondence to: Lewis Hsu, MD, PhD, Emory University School of Medicine, 69 Butler St SE, Atlanta, GA 30303
92S
Thomas L. Petty 41st Annual Aspen Lung Conference: Acute Lung Injury