Claus Danckert Krohn

The Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway Is Activated by Lipoteichoic Acid and Plays a Role in Kupffer Cell Production of Interleukin-6 (IL-6) and IL-10

ABSTRACT Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-α levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-α, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.

Complement activation and release of interleukin‐6 and tumour necrosis factor‐α in drained and systemic blood after major orthopaedic surgery

OBJECTIVE To measure the concentration of complement activation products C3bc and terminal complement complex (TCC) and the cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) in systemic and drained wound blood for the first six hours after a major orthopaedic operation. DESIGN Prospective study. SETTING University hospital, Norway. PATIENTS 8 patients operated on for thoracic scoliosis. MAIN OUTCOME MEASURE Concentrations of complement activation products, IL-6 and TNF-alpha in arterial (systemic) and drained (local) blood were measured at wound closure and 1, 2, 4, and 6 hours postoperatively. RESULTS C3bc and TCC were 10 times higher, and IL-6 showed extreme values, in drained compared with arterial blood. The concentration of TNF-alpha did not increase significantly, either in drained or in arterial blood. The white cell count (WCC) increased threefold in both drained and arterial blood compared with arterial control values before operation. CONCLUSION Complement activation and IL-6 release after surgical trauma differ significantly in local and systemic blood samples. Conclusions based only on systemic findings may be limited.

The cytokines IL-1β and IL-1 receptor antagonist, IL-2 and IL-2 soluble receptor-α, IL-6 and IL-6 soluble receptor, TNF-α and tnf soluble receptor I, and IL10 in drained and systemic blood after major orthopaedic surgery

Objective: To measure the concentration of the cytokines interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumour necrosis factor-α (TNF-α) and the modulators of their function interleukin-1 receptor antagonist (IL-1Ra), interleukin-2 soluble receptor alpha (IL-2 sRα), interleukin-6 soluble receptor (IL-6sR) and soluble tumour necrosis factor receptor I (sTNFR-I) in systemic and drained blood for the first six hours after a major orthopaedic operation.Design: Prospective study.Setting: University hospital, Oslo.Patients: 8 patients operated on for thoracic scoliosis.Main outcome measure: Concentrations of IL-1β, IL-2, IL-6, IL-10, TNF-α, IL-1Ra, IL-2 sRα, IL-6sR, and sTNFR-I were measured together with haemoglobin (Hb) concentration, white cell count (WCC), and differential count in arterial and drained blood at wound closure and 1, 2, 4, and 6 hours postoperatively.Results: IL-1β and IL-6 concentrations increased significantly in drained blood, whereas that o...

Fibrinolytic activity and postoperative salvaged untreated blood for autologous transfusion in major orthopaedic surgery

OBJECTIVE To evaluate the fibrinolytic activity in a closed surgical wound, in postoperatively drained blood, and during autologous transfusion. DESIGN Prospective study. SETTING National hospital, Norway. PATIENTS 9 patients operated on for thoracic scoliosis. MAIN OUTCOME MEASURE Concentrations of plasmin/antiplasmin (PAP), alpha2-antiplasmin, and D-dimers in drained, arterial, and mixed venous blood before, during, and after infusion of 10 ml/kg body weight of postoperatively drained, untreated blood. RESULTS In drained blood the concentration of alpha2-antiplasmin was 31% of the preoperative arterial control value. Together with the increased concentrations of PAP to 18076 microg/L and D-dimers to 126 mg/L, this indicates extensive fibrinolytic activity in the closed wound. The postoperative autologous transfusion of drained, untreated blood increased the concentration of PAP from 507 to 2453 microg/L and of D-dimer from 0.7 mg/L to 15.3 mg/L in systemic blood. CONCLUSION The systemic concentration of fibrin(ogen) degradation products, indicated by D-dimers, after recirculation of drained, untreated blood might impair coagulation. The extensive activation of plasmin might exhaust available alpha2-antiplasmin in the wound and result in postoperative rebleeding.

Impact of hypertonic saline on the release of selected cytokines after stimulation with LPS or peptidoglycan in ex vivo whole blood from healthy humans.

The question of specific immunomodulating qualities of hypertonic saline (HTS) has not been settled. It has proven difficult to distinguish between immunomodulation directly attributable to HTS and influence because of favorable circulatory effects. The nature of immune activator may also play a role. In a whole-blood model, we have investigated these relations further, with special emphasize on osmolalities usually found after recommended dosing. Blood from 10 healthy donors was exposed to osmolalities ranging from 295 to 480 mOsm/kg and stimulated with the two clinically relevant stimulators peptidoglycan (1 &mgr;g/mL) or LPS (10 ng/mL) for 6 h at 37°C. Leukocyte response was evaluated by measuring selected cytokines in the supernatant. Moderate hyperosmolality alone boosted the release of CXCL8/IL-8. The peptidoglycan-stimulated synthesis of pivotal proinflammatory cytokines was inhibited in an osmolality-dependent way, but statistically significant only at osmolalities above those attained after routine use of HTS, i.e., 310 mOsm/kg or greater: IL-6 (P < 0.05 at 315 mOsm/kg), IL-1&bgr;, and TNF-&agr; (P < 0.05 at 335 mOsm/kg). Similar effects were seen for the chemokine CCL3 and the anti-inflammatory cytokine IL-10. In contrast, the effects in cells stimulated with LPS were either lower or absent. Thus, osmolalities usually found after clinical use of HTS only modestly influenced the selected immune parameters, regardless of stimulator.

Cytokine Responses to Glucocorticoids and Surgery

Purpose:This prospective randomized study was designed to evaluate the implication of high preoperative doses of glucocorticoids on the cytokine responses after surgical correction in patients with ankylosing spondylitis.Patients and Methods:In 20 consecutive patients an extending osteotomy of the lumbar spine was done by a wedge excision. In a random manner, 10 of the patients were given 10 mg/kg of methylprednisolone (“Solum-Medrol”, Pharmacia & Upjohn, Stockholm, Sweden) preoperatively. The control patients received the same amount of saline. Arterial blood was sampled before and at the end of operation and at 4 and 24 h postoperatively and analyzed for pro- and antiinflammatory cytokines.Results:Surgery induced non-significant increases in TNF-α and IL-1-β and significant increases in IL-6, IL-8, IL-10 and sTNF-R1 in both patient groups. Glucocorticoids significantly reduced increases in IL-6 and IL-8. On the other hand, increases in IL-10 and sTNF-R1 were significantly enhanced by corticoids. In both groups CRP was significantly increased at 24 h after surgery, but the increments were significantly reduced by corticoids.Conclusion:This study shows that surgery adds a complexity to cytokine productions in patients with ankylosing spondylitis, and that the balance of these cytokines is significantly influenced by glucocorticoids.

Effects of Infliximab and Hydrocortisone on In Vitro Cytokine Responses after Stimulation with Lipopolysaccharide

BACKGROUND Both glucocorticosteroids and biologic drugs such as the tumor necrosis factor (TNF)-α antagonist infliximab are used often in the treatment of rheumatoid arthritis or inflammatory bowel disease. In severe disease, or if allergic reactions occur during treatment with infliximab, combined therapy with these drugs often is instituted. Combining infliximab and glucocorticosteroids may increase substantially the risk of severe opportunistic infections or dissemination of malignant tumors because of their additive effects as immunosuppressants. METHODS In a whole-blood in vitro model, we studied the influence of different doses of infliximab and hydrocortisone, either separately or in combination, on the synthesis of selected cytokines after stimulation with lipopolysaccharide (LPS). RESULTS Hydrocortisone in therapeutic serum concentrations significantly inhibited the expression of a majority of the cytokines tested. Infliximab, in serum concentrations relevant to clinical situations, inhibited TNF-α activity significantly. This effect was potentiated when infliximab was combined with hydrocortisone. Similar effects were found using a low dose of infliximab combined with hydrocortisone. Infliximab alone inhibited the expression of the cytokines interleukin (IL)-1 receptor antagonist, monocyte chemoattractant protein-1, IL-8, and IL-12. Hydrocortisone in combination with low-dose infliximab potentiated the suppressive effects on TNF-α, IL-1β, IL-8, and macrophage inflammatory protein-1α synthesis. CONCLUSIONS Immune-modulating effects of infliximab were found both in clinically relevant doses and, most notably, in low doses reflecting serum concentrations found commonly in patients several months after the last injection. Infliximab potentiates the suppressive effects of hydrocortisone on cytokine synthesis.

Systemic and local cytokine kinetics in musculoskeletal injury : a prospective study in patients with ankylosing spondylitis

Objective. Back surgery in patients with ankylosing spondylitis is a major trauma in individuals with tissue inflammation and joint destruction along the spine; we used surgery in these patients as a model in the study of systemic and local cytokine profiles in complicated trauma situations. Material and methods. Blood was sampled before, during and after surgery in 10 patients operated on with extending osteotomy of the lumbar spine. Samples of arterial blood and local wound blood were analysed for proinflammatory and anti‐inflammatory cytokines. Results. Surgery induced no significant changes in systemic values of TNFα and IL‐1β. There were significant increments in systemic values of IL‐6, IL‐8 and sTNF‐R1. A systemic increase in values of IL‐10 was only noticed after 24 h. There were increments in local values of TNFα at 24 h and in local values of IL‐1β, IL‐6, Il‐8 and IL‐10 at both 4 and 24 h postoperatively. The local values were in general significantly higher than the systemic values. Conclusions. This study indicates that a major musculoskeletal trauma principally is followed by significant increases in systemic levels of IL‐6 with only modest systemic reactions in TNFα and IL‐1β, even in patients with an inflammatory disease. However, there are in general significantly increased local levels of IL‐1β, IL‐6, IL‐8 and IL‐10, and our conclusion is that systemic cytokine levels might not reflect local reactions.