Adult respiratory distress syndrome (ARDS) results from acute lung injury and carries a mortality rate in excess of 60%. If ARDS is complicated by bacterial sepsis, mortality approaches 90%. Gram negative bacteria produce lipopolysaccharide endotoxin (LPS), known to cause macrophages to secrete cytokines such as tumor necrosis factor (TNF) and interleukin 1 (IL-1). Intravenous injection of minute quantities of LPS or TNF can reproduce most of the overwhelming systemic effects of septic shock, including diffuse pulmonary inflammation and edema. Thus, LPS and macrophage-derived cytokines are likely to play key roles in the progression from acute lung injury to full-blown ARDS. Accordingly, an understanding of the mechanisms by which these cytokines are regulated, and the impact of LPS on that regulation, should aid in the design of therapeutic strategies to prevent the development of ARDS in patients who have sustained acute lung injuries. We have discovered that in cultured monocytes and macrophages, TNF mRNA that has been induced by another cytokine (granulocyte-macrophage colony stimulating factor, GM-CSF) fails to be translated into protein until a second signal, LPS, is supplied. This effect is specific for the TNF mRNA. We have observed that the cells require both LPS and the expression of an additional gene, before translation of the TNF mRNA can proceed. We propose to dissect the mechanism of translational regulation of TNF gene expression, since understanding how the TNF gene is normally controlled, and how LPS disrupts this, could greatly enhance the development of the TNF gene is normally controlled, and how LPS disrupts this, could greatly enhance the development of treatment strategies for patients at risk for developing ARDS.
Our specific aims are designed to determine the molecular basis of our observation that GM-CSF-induced TNF mRNA is translationally inhibited, and that LPS can override this control. 1. We will characterize the 5' cap structure and poly(A) tail of TNF mRNA that is induced by GM-CSF. 2. We will characterize the macrophage cytoplasmic protein responsible for inhibition of translation of TNF mRNA, and clone the DNA that encodes it. 3. We will clone the gene that is induced by LPS exposure and required for translation of pre-existing TNF mRNA. 4. We will analyze TNF mRNA induced by other stimuli, to identify other situations in which TNF gene regulation includes translational control, and to determine whether LPS deregulates these mechanisms as well. Our ultimate goal is to understand this mechanism well enough to facilitate the design of specific therapies to assist patients in whom inflammatory cytokine overproduction contributes to serious illness.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL046966-03
Application #
3473605
Study Section
Pathology A Study Section (PTHA)
Project Start
1991-08-01
Project End
1996-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Ryan, L K; Golenbock, D T; Wu, J et al. (1997) Characterization of proinflammatory cytokine production and CD14 expression by murine alveolar macrophage cell lines. In Vitro Cell Dev Biol Anim 33:647-53
Ogata, M; Fletcher, M F; Kloczewiak, M et al. (1997) Effect of anticoagulants on binding and neutralization of lipopolysaccharide by the peptide immunoglobulin conjugate CAP18(106-138)-immunoglobulin G in whole blood. Infect Immun 65:2160-7
Herlihy, J P; Vermeulen, M W; Hales, C A (1996) Human alveolar macrophages prevent apoptosis in polymorphonuclear leukocytes. Am J Physiol 271:L681-7
Ryan, L K; Vermeulen, M W (1995) Alveolar macrophages from C3H/HeJ mice show sensitivity to endotoxin. Am J Respir Cell Mol Biol 12:540-6
Polanski, M; Vermeulen, M W; Wu, J et al. (1995) Muramyl dipeptide mimicry in the regulation of murine macrophage activation by serotonin. Int J Immunopharmacol 17:225-32