Cynthia Mendez

Specific pancreatic enzymes activate macrophages to produce tumor necrosis factor-alpha: role of nuclear factor kappa B and inhibitory kappa B proteins.

The triggering events by which mononuclear cells throughout the body are induced to produce large amounts of cytokines during acute pancreatitis are unclear. However, recent work in our laboratory demonstrated that three specific pancreatic enzymes (elastase, carboxypeptidase A, and lipase) induced dramatic tumor necrosis factor-alpha (TNF-α) protein production from macrophages, whereas all others could not. This series of experiments was designed to examine the second messenger system by which this occurs. The rat macrophage cell line NR8383 was incubated for 3 hours with elastase, carboxypeptidase A, lipase, trypsin, or lipopolysaccharide (positive control). Activation of nuclear factor kappa B (NF-KB) was demonstrated by electrophoretic mobility shift assay, presence of inhibitory kappa B alpha and beta (IKB-a and IKB-13) by Western ~lot analysis, and TNF-α protein production by enzyme-linKed immunosorbent assay. Elastase, carboxypeptidase A, and lipase induced degradation of IKB-13 (but not IKB-a), activation of NF-KB, and production of TNF-α protein, whereas inhibition of IKB with pyrrolidine dithiocarbamate attenuated this response. Trypsin was unable to elicit any of these responses. Macrophages can be induced by specific activated pancreatic enzymes-elastase, carboxypeptidase A, and lipase--to produce TNF-α. This process is dependent on IKB-13 degradation and NF-KB activation, suggesting that these enzymes trigger this second messenger system through specific membrane-bound receptors.

Sublethal hemorrhage induces tolerance in animals exposed to cecal ligation and puncture by altering p38, p44/42, and SAPK/JNK MAP kinase activation.

BACKGROUND We have shown that SLH induces tolerance to endotoxin in vivo and in vitro, and is associated with alterations in MAP kinase (p38, p44/42, and SAPK/JNK) activation and TNF production. This study investigates the effect of sublethal hemorrhage (SLH) on cecal ligation and puncture (CLP) polymicrobial sepsis and examined the effect of the bioflavinoid, curcumin, a MAP kinase inhibitor, on this relationship. MATERIALS AND METHODS Sprague-Dawley rats underwent SLH (hemorrhage and MAP = 30 mm Hg for 15 min, with shed blood returned) or sham operation. After 24 h, rats had CLP (cecal base ligation with double puncture). Survival was determined +/- curcumin pretreatment (n = 10/group). Lung tissue, serum, and bronchoalveolar lavage (BAL) fluid were obtained 30 min after SLH and 4 and 12 h after CLP (n = 8/group). Lung tissue was analyzed for p38, p44/42 SAPK/JNK, and HSP-70 phosphorylation (Western). Lung myeloperoxidase (MPO) activity was measured as an index of neutrophil infiltration. TNF ELISA was performed on serum and BAL sample. RESULTS SLH significantly improved survival after CLP (21.5 vs. 7.5 h vs. sham, p = 0.008), and curcumin reversed this benefit (7.3 h, p = 0.0007 vs. SLH + CLP). MAP kinase activity was significantly greater in SLH rats 4 h post-CLP (p38: 720 vs. 331, p = 0.03, p44/42: 2759 vs. 1295, p = 0.006, SAPK: 413 vs. 254). Curcumin significantly inhibited MAPK activity both 30 min after SLH (p38: 297 vs. 3260, p44/42: 370 vs. 2628, SAPK: 748 vs. 1764, all p < 0.01 vs. SLH 30 min) and 4 h post CLP (p38: 146 vs. 720, p44/42: 616 vs. 2759, all p < 0.01 vs. SLH + CLP4 h). Four hours after CLP, SLH rats expressed more HSP72. Lung MPO activity was significantly lower in SLH + CLP rats at both 4 h (9.5 vs. 15.6, p = 0.02 vs. sham) and 12 h (18.1 vs. 37.5, p = 7 x 10(-5), vs. sham). Serum and BAL TNF levels were higher in SLH rats initially (serum: 145 vs. 28 pg/mL, p = 2 x 10(-5) BAL: 83 vs. 57 vs. sham + CLP4h); however, BAL TNF was significantly lower in SLH animals 12 h post-CLP (37 vs. 72.7 pg/mL, p = 0.003 vs. sham + CLP12h). CONCLUSION SLH induces tolerance to CLP. This tolerance is dependent on early MAP kinase activation, since the survival benefit is reversed by curcumin. Decreases in tissue cytokine levels and neutrophil infiltration are potential mechanisms by which SLH induces tolerance to CLP (polymicrobial sepsis), attenuates acute lung injury, and improves survival.

Effects of tolerizing sublethal hemorrhage on p44/42 and SAPK/JNK Map-kinase activation.

Exposure to sublethal hemorrhage (SLH) makes rats tolerant to subsequent hemorrhagic or septic shock. We have shown that this tolerance leads to alterations in cytokine production, macrophage NF-&kgr;B activation and p38 MAP-kinase activity. The purpose of this study was to explore whether changes in p44/42 and SAPK/JNK MAP kinase activity also occur after the induction of tolerance by SLH. Rats were made tolerant by SLH (mean arterial pressure = 30 mmHg for 15 min with shed blood returned). Shams had anesthesia and instrumentation only. Twenty-four hours after SLH or sham operation, LPS was given (40 mg/kg intraperitoneal). Lung, liver, and splenic tissues were harvested 15, 30, and 45 min following sham, SLH, or LPS. Protein was isolated from tissues for determination of p44/42 and SAPK/JNK phosphorylation by Western blot analysis. Phosphorylation of p44/42 and SAPK/JNK was detected in all tissues following both sham and SLH, and this effect was significantly more pronounced following SLH (P < 0.05). However, activation of both p44/42 and SAPK/JNK in response to LPS, was significantly lower in the SLH rats when compared to shams. Peak activation was seen 30 min after SLH and peak attenuation, 30 min after LPS. The amount of nonphosphorylated protein was comparable in all groups. The induction of tolerance by SLH leads to phosphorylation of both p44/42 and SAPK/JNK MAP-kinases. However, the activation of these same kinases is attenuated in response to LPS in animals made tolerant by SLH.

Heat shock protein (HSP72) and p38 MAPK involvement in sublethal hemorrhage (SLH)-induced tolerance.

BACKGROUND Our in vivo model of tolerance, sublethal hemorrhage (SLH), alters cytokine production, nuclear factor-kappaB mobilization, mitogen-activated protein (MAP) kinase activity, and makes rats tolerant to shock. Heat shock protein (HSP) protects animals from stress. This study investigated if SLH induces in vivo HSP72 expression and whether in vitro HSP72 induction by sodium arsenite (NaArs) alters intracellular signal transduction and cytokine production similar to SLH. METHODS Sprague-Dawley rats were made tolerant by SLH (MAP = 30 mmHg for 15 min, shed blood returned) and given lipopolysaccharide (LPS; 40 mg/kg i.p.) 24 h later. Lung was harvested 1, 12, and 24 h after SLH (n = 4) and 1 h after LPS (n = 8). Other rats underwent bronchoalveolar lavage 24 h after SLH, and macrophages (mphi) were treated with LPS (10 microg/ml). The NR8383 alveolar mphi cell line was treated with 50 microM NaArsx 12 h and LPS. Reverse transcription polymerase chain reaction, Western blots, and enzyme-linked immunosorbent assay were performed for gene, MAPK, and protein expression (tumor necrosis factor [TNF], HSP, p38). RESULTS SLH induced significantly more lung HSP72 mRNA and protein. SLH mphi had more HSP72 protein before and after LPS compared with shams. NaArs induced HSP72 mRNA and protein in NR8383 mphi, and these cells made less TNF compared with controls. NaArs significantly increased p38 activation vs control. SB203580 inhibition of p38 activity did not affect HSP72 expression, or reverse NaArs inhibition of LPS induced TNF production. CONCLUSION SLH induces HSP72 in vivo. In vitro HSP72 induction is associated with increased p38 phosphorylation. Like SLH, mphi with induced HSP72 expression, have an attenuated TNF response. HSP72 acts independently from p38 in inducing tolerance.