Impaired inducibility of heat shock protein 70 in peripheral blood lymphocytes of patients with severe sepsis.

OBJECTIVE: To determine the extent of the potentially protective heat shock protein 70 response in peripheral blood lymphocytes of patients with severe sepsis after ex vivo lipopolysaccharide stimulation. DESIGN: Entry study of consecutive patients with severe sepsis, those who were critically ill or nonseptic after major surgery, and healthy blood donors. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Ten patients with diagnoses of severe sepsis; ten critically ill, nonseptic patients after major surgery; and ten healthy blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We investigated the ex vivo endotoxin-inducible expression of heat shock protein 70 in peripheral blood lymphocytes of patients with severe sepsis by means of flow cytometry. Only negligible amounts of inducible intracellular heat shock protein 70 accumulation (<4.2% of lymphocytes) could be detected in peripheral blood lymphocytes without lipopolysaccharide stimulation. The proportion of cells accumulating heat shock protein 70 after treatment with lipopolysaccharide was distinctly lower in patients with severe sepsis (p < .05) than in critically ill, nonseptic patients after major surgery and healthy blood donors (38.3+/-3.3%, 82.2+/-4.5%, and 70.9+/-3.9%, respectively; mean +/- SEM; n = 10). Patients with clinical signs of recovery from severe sepsis showed an increase in heat shock protein 70 expression. CONCLUSIONS: Inducibility of ex vivo heat shock protein 70 was impaired in peripheral blood lymphocytes of patients with severe sepsis. The impaired expression of the potentially protective heat shock protein 70 may contribute in vivo to immune dysfunction, because intact functioning of T and B lymphocyte responses is of central importance in resisting infection in severe sepsis. Monitoring of inducible heat shock protein 70 in peripheral blood lymphocytes may contribute to the evaluation of the immune consequences of severe sepsis.

Surgical stress induces a shift in the type-1/type-2 T-helper cell balance, suggesting down-regulation of cell-mediated and up-regulation of antibody-mediated immunity commensurate to the trauma.

BACKGROUND: Measuring serum cytokines, pituitary hormones, or acute phase proteins during or after surgery is not an optimal method for quantifying the impact of surgical procedures. In an effort to assess surgical stress by means of the immune response, we focused on changes in cell-mediated and antibody-mediated immunity as illustrated by the type 1/type 2 T-helper (Th1/Th2) cell balance. The sensitivity of this approach was evaluated by comparing laparoscopic and conventional cholecystectomy (LCE, CCE). METHODS: In a pragmatic prospective study 43 patients with symptomatic cholelithiasis were operated on either by LCE (n = 25) or CCe (n = 18). Blood sampling was done 24 hours before surgery, immediately before incision, and 2, 24, and 48 hours after surgery. Cell surface markers and cytokine production were used to characterize the Th1/Th2 balance and were measured by means of flow cytometry and enzyme-linked immunosorbent assay techniques. RESULTS: Activation of Th2 cells evokes the production and secretion of interleukin-4 (IL-4), which up-regulates the expression of immunoglobulin E receptors (Fo epsilon RII, CD23) on B cells. Phytohemagglutinin-induced IL-4 production in freshly isolated peripheral blood mononuclear cells from patients increased more after CCE than LCE (IL-4, +41% versus +17%; p < 0.05). Also the expression of CD23 on B cells was higher after CCE than LCE (+146% versus +63%; P < 0.01). CD30, a membrane molecule that belongs to the tumor necrosis factor receptor superfamily and probably is an important indicator of Th2 activity, was more evaluated on T cells from patients who underwent CCE. The Th1 response, characterized by phytohemagglutinin-induced IFN-gamma secretion in peripheral blood mononuclear cells and up-regulation of human leukocyte antigen-DR expression on monocytes, was lower after CCE than after LCE. CONCLUSIONS: This study shows that surgical stress induces a shift in the Th1/Th2 balance toward Th2, suggesting that cell-mediated immunity is down-regulated and antibody-mediated immunity is up-regulated after surgery. The evaluation of this shift may be clinically meaningful and help quantify even less invasive surgical procedures. When comparing CCE and LCE in this not strictly randomized study, we found LCE to be the less stressful procedure.

Protein kinase C regulates IL-8 and fMLP induced cytoplasmic Ca2+ increase in human granulocytes by receptor modulation measurements by flow cytometry.

Changes in cytosolic free Ca2+ influence important granulocyte functions like chemotactic behavior, adherence to endothelia, and phagocytosis. In the following study we used a simple reproducible procedure involving flow cytometry in combination with the fluorescent dye Fluo-3 to measure Ca2+ changes in human granulocytes. The aim of our study was to investigate the involvement of protein kinase C in regulating cytosolic free Ca2+ concentrations after stimulation of cells with IL-8 and fMLP. Both reagents induced a 5-6 fold increase in cytosolic Ca2+. Experiments conducted in Ca(2+)-free media showed a minor 18-29% decrease in cytosolic Ca2+ response, suggesting that intracellular Ca(2+)-stores are the main source for Ca2+ release after fMLP or IL-8 stimulation. Activators of protein kinase C, phorbol myristate acetate (PMA) and 1-oleyl-2-acetyl-sn-glycerol (OAG), inhibited cytosolic Ca(2+)-increase completely when induced by IL-8 and by 68-82% in the case of fMLP. Staurosporine, an inhibitor of protein kinase C, was able to attenuate or even abolish the PMA/OAG-effect. Our results show that changes in cytosolic Ca2+ due to IL-8 and fMLP signalling can be regulated by protein kinase C in human granulocytes. This regulatory role of protein kinase C involves some form of receptor modulation (i.e. phosphorylation, internalization, shedding).

Atrial natriuretic peptide protects hepatocytes against damage induced by hypoxia and reactive oxygen. Possible role of intracellular free ionized calcium.

Elevated concentrations of atrial natriuretic peptide reportedly mitigate acute renal failure in vivo and in the isolated perfused kidney (M. Nakamoto, J.I. Shapiro, P.F. Shanley, L. Chan & R.W. Shrier (1987) J. Clin. Invest. 80, 698-705; S.G. Shaw, J. Weidmann, J. Hodler, A. Zimmermann & A. Paternostro (1987) J. Clin. Invest. 80, 1232-1237). Since atrial natriuretic peptide has been shown to be a potent vasodilator, this beneficial effect may be due entirely to improved haemodynamics. To determine whether atrial natriuretic peptide also has a protective effect at the cellular level, rat hepatocyte cell cultures were treated with atrial natriuretic peptide prior to or after induction of cell damage by hypoxia (0.5% O2 for 4 h) or reactive oxygen (hypochlorous acid). Bleb formation, degradation of radiolabeled trichloroacetic acid-precipitable peptides, release of lactate dehydrogenase and trypan blue exclusion were used as indicators of cell damage. Atrial natriuretic peptide treatment distinctly protected the cell cultures against damage in both cases. This beneficial effect of atrial natriuretic peptide was partly mimicked by sodium nitroprusside, which, like atrial natriuretic peptide, largely increased the cellular cGMP content. 6-Anilino-5,8-quinolinedione (Ly 83583), an inhibitor of particulate guanylate cyclase, blocked the protective effect of atrial natriuretic peptide. Therefore a cGMP-mediated mechanism seems to be involved in the cytoprotective action of atrial natriuretic peptide. Fluorometric measurements using the Ca2+-sensitive dye Quin-2 showed that the elevation of intracellular Ca2+ after cellular insult by hypochlorous acid is prevented by atrial natriuretic peptide. These results suggest that atrial natriuretic peptide may attenuate hypoxic and toxic cell damage by increasing cGMP and reducing intracellular Ca2+.

Protein kinase C involvement in lipid peroxidation and cell membrane damage induced by oxygen-based radicals in hepatocytes.

We investigated the effects of oxygen-based radicals induced by t-butyl hydroperoxide or H2O2/Cu2+ on cultured hepatocytes. Radical exposure caused membrane lesions (blebs), lactate dehydrogenase release and lipid peroxidation (i.e. formation of malondialdehyde) in cells. As expected, radical scavengers (catalase, alpha-tocopherol) strongly inhibited these phenomena. A similar or even superior inhibitory effect was achieved by the protein kinase C (PKC) inhibitors H-7 and phloretin. These agents did not reveal notable radical scavenging properties as assessed by their ability to break down H2O2. The PKC stimulators 4 beta-phorbol-12-myristate-13 and 1-olyeoyl-2-acetyl-sn-glycerol intensified the detrimental actions of the radical-inducing agents. [3H]Phorbol-12,13-dibutyrate-binding studies showed that membrane association of PKC is markedly increased in hepatocytes after exposure to H2O2/Cu2+ or t-butyl hydroperoxide. These results suggest that PKC membrane translocation and activation may be important for mediating membrane damage and lipid peroxidation after cells are exposed to oxygen-based radicals.

Calcium metabolism and cystic fibrosis: mitochondrial abnormalities suggest a modification of the mitochondrial membrane.

A disorder of calcium (Ca2+) metabolism may be central to the pathogenesis of cystic fibrosis (CF). Average cellular Ca2+ levels in fibroblasts derived from patients with CF (ages, 14-25 yr; n = 25) were 36-77% higher than in matched controls depending on age of cell culture (9.0-10.6 versus 5.1-7.8 nmol/mg cellular protein). Cellular Ca2+ was significantly elevated in CF, but was not a reliable criterion for identifying CF cells because of the high variability of results. Studies of Ca2+ fluxes in cell organelles showed that mitochondria isolated from CF fibroblasts accumulate 2-3 times more Ca2+ than controls [79.5 +/- 8.2 versus 33.7 +/- 4.7 nmols X mg mitochondrial protein-1 X 10 min-1 (+/- SD)], Ca2+ accumulation in mitochondrial reliably distinguished between CF and control or heterozygote cells (P less than 0.0005, n = 11). In vitro experiments showed that Ca2+ influx and efflux are increased in isolated CF mitochondria, resulting in net Ca2+ accumulation. Ca2+ uptake in mitochondria is energy-dependent; some inhibitors of mitochondrial energy metabolism (atractyloside, oligomycin) influenced Ca2+ intake significantly more in CF than in control mitochondria. Furthermore, the average activities of NADH oxidase, NADH- and succinate-cytochrome c reductase were 77, 58, and 48% higher in CF mitochondria, respectively. This indicates that many functions associated with energy metabolism and the mitochondrial membrane (electron transport, ATP transport, and ATP hydrolysis) are not operating properly in CF, thus possibly causing the derangement of Ca2+ metabolism found in CF mitochondria and cells.