Hemodynamic variables and plasma levels of PGI2, TXA2 and IL-6 in a porcine model of recurrent endotoxemia.

The pulmonary and systemic hemodynamic effects of recurrent endotoxemia were studied in pigs over a 48-hr period. Six pigs of the test group were given 0.5 micrograms/kg of an E. coli endotoxin (WO111: B4) over 60 min at the beginning and in the middle (22 hr) of the experiment. Three pigs given the same amount of physiological saline solution served as controls. The hemodynamic response to the first LPS injection was characterized by severe pulmonary hypertension, a significant increase in systemic vascular resistance, and a marked decrease in cardiac output. Circulating TxB2 levels were higher than those of 6-keto-PGF1 alpha levels, so that the first response to LPS is influenced by the vasoconstrictive actions of TxA2. With the second LPS application, the pulmonary response was attenuated, although a significant increase of pulmonary artery pressure and pulmonary vascular resistance occurred. Once again systemic vascular resistance rose and cardiac output decreased, but this time plasma levels of 6-keto-PGF1 alpha were greater than those of TxB2. Toward the end of the experiment, we noted the progressive onset of a hyperdynamic and hypotensive state. Systemic vascular resistant index decreased to 50% of the baseline value. IL-6, a cytokine of systemic importance during the course of septic shock, markedly and significantly peaked after each LPS injection. Circulating plasma levels in response to recurrent endotoxemia are described.

The three carboxy-terminal amino acids of human interleukin-6 are essential for its biological activity.

We have constructed on the cDNA level deletion mutants of human interleukin-6 lacking one, two, three or four amino acids from the carboxy-terminus of the molecule. After in vitro transcription and translation the biological activity of these deletion mutants was determined by two independent bioassays. Both, the mouse B9 cell proliferation assay and the fibrinogen induction assay with the human hepatoma cell line HepG2 led to the following result: already the removal of the last amino acid resulted in a five-fold loss of biological activity. An additional slight reduction was seen when two amino acids were removed from the carboxy-terminus. Interleukin-6 lacking three or four C-terminal amino acids were completely inactive. The presented results emphasize the extreme importance of the carboxy-terminus of interleukin-6 for its biological function.

Studies on the structure and regulation of the human hepatic interleukin-6 receptor.

Affinity cross-linking of 125I-labeled recombinant human interleukin-6 (IL-6) to human hepatoma cells (HepG2) allowed the detection of three IL-6-containing complexes with molecular masses of 100 kDa, 120 kDa and 200 kDa. Treatment of HepG2 cells with dexamethasone led to a time- and dose-dependent up-regulation of IL-6-receptor mRNA levels. By the use of cross-linking this effect was also seen at the protein level, where all three IL-6-binding complexes increased upon incubation of HepG2 cells with dexamethasone. Under conditions of IL-6-receptor up-regulation by dexamethasone, gamma-fibrinogen mRNA induction by IL-6 is stronger and occurs earlier than without dexamethasone. We propose therefore that the expression of the IL-6 receptor might be a rate-limiting step in acute-phase-protein induction.

Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes.

Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis.

Microheterogeneity of human interleukin 6 synthesized by transfected NIH/3T3 cells: comparison with human monocytes, fibroblasts and endothelial cells.

A cDNA containing the entire coding region of human interleukin 6 (IL 6) was stably expressed in murine NIH/3T3 fibroblasts using a bovine papilloma virus-based expression vector with a metallothionein promoter. Expression of IL 6 in transfected cells was highly inducible by heavy metals like cadmium as measured at mRNA and protein levels. Cadmium-stimulated transfected NIH/3T3 cells synthesized and exported biologically active IL 6 (1.7 x 10(4) U/10(6) cells/24 h). IL 6 from the culture medium of transfected NIH/3T3 cells exhibited at least eight bands on Western blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that human IL 6 expressed in NIH/3T3 cells shows a complex glycosylation pattern. Using glycosidases and the N-glycosylation inhibitor tunicamycin it was possible to discriminate between five species carrying N-linked carbohydrate side chains and two species carrying only O-linked side chains. In addition, a substantial amount of unglycosylated IL 5 was observed. IL 6 from transfected NIH/3T3 cells differed markedly in its glycosylation pattern from those of stimulated human monocytes, fibroblasts and endothelial cells.

Activation of bone marrow-derived mouse macrophages by bacterial lipopeptide: cytokine production, phagocytosis and Ia expression.

The lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-a lanyl-glycine (Pam3Cys-Ala-Gly), a synthetic analogue of the N-terminal part of bacterial lipoprotein, induces the secretion of interleukin (IL) 1, IL 6 and tumor necrosis factor (TNF)-alpha in bone marrow-derived macrophages that have been cultured in vitro for up to 20 days. IL 6 and TNF-alpha secretion increased from day 6 to day 20 whereas IL 1 secretion increased until day 13 and decreased on day 20. In contrast to the enhancement of cytokine production, phagocytosis of IgG-coated sheep erythrocytes and Ia expression were found to be diminished after treatment with lipopeptide for 24 h. Morphological studies revealed lipopeptide-induced changes of macrophage cultures. The data presented here show the potential of the lipopeptide as a strong activator of bone marrow-derived macrophages.

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.