Influence of Low Molecular Weight Heparin (Certoparin) and Unfractionated Heparin on the Release of Cytokines from Human Leukocytes

We analyzed the influence of heparins (unfractionated heparin, UFH and low molecular weight heparin certoparin) on the generation of IL-1ra, IL-6, IL-10, and IL-12p40 and from leukocyte fractions in vitro. Polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from 16 different healthy donors were isolated and adjusted to 1 × 10⁶ cells/ml supplemented RPMI 1640. Leukocyte fractions were differentially stimulated (PMN with 1 g and 5 g LPS, PBMC with 10 ng TSST-1 or 2 g ConA) in the presence or absence of heparins (1 U/ml, 2 U/ml, and 4 U/ml) for 24 h at 37°C. Cytokine release was analyzed by ELISA. Certoparin but not UFH led to a dose-dependent increase in IL-6 from non-stimulated PBMC. In contrast, the release of IL-1ra, IL-10, and IL-12p40 was not modulated by heparins in a dose-dependent fashion. Increases in these cytokines occurred only as single incidents at intermediate heparin levels. An influence of the heparins on the apoptosis of PMN (measured as DNA-fragmentation in non-stimulated or LPS-stimulated cell-fractions) was not observed.     

Intravenous immunoglobulin induces interferon-γ and interleukin-6in vivo

Immunoglobulin is known to be an immunomodulator. It can induce protein mediators from mononuclear cells, particularly monocytesin vitro. Intravenous immunoglobulin (IVIg) has been used as a therapy in several clinical situations. In this study, the influence of IVIg infusion on the plasma levels of two protein mediators, interferon- (IFN-) and interleukin-6 (IL-6), was assessed in patients with secondary generalized epilepsy. Compared to preinfusion levels, plasma interferon- was increased in 18 of 18 patients 20 min after the 6- to 8-hr infusion of IVIg. Plasma interferon- levels reached their peak at various times from 20 min to 3 days post IVIg infusion, dependent upon the individual patient. Plasma IL-6 levels also increased after IVIg infusion. Generally, IL-6 reached its peak level after IFN-. No activated T cells or B cells were observed as determined by the expression of surface CD25, CD23, and HLA-DR 20 min following the infusion when the IFN- and IL-6 levels were assessed. The expression of the high-affinity receptor for IgG, CD64, on monocytes was significantly enhanced after IVIg infusion, while the low-affinity receptor for IgG, CD32, was only slightly increased. Cytoplasmic staining of PBMC indicates that both CD16-positive and CD16-negative cells may contribute to the increase seen in plasma IFN-. These data raise the possibility that the therapeutic effects of intravenous immunoglobulin may be related, at least in part, to the immunomodulatory activity as demonstrated by the changes in plasma levels of IFN- and IL-6.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Neuropeptide regulation of interleukin-1 activities

-Melanocyte stimulating hormone (-MSH), a 13 amino acid neuropeptide produced by the pituitary gland, was found to markedly inhibit the capacity of exogenously administered interleukin-1 (IL-1) to stimulate the enhanced synthesis of acute-phase proteins and induce neutrophilia in vivo. The administration of ACTH or glucocorticosteroids lacked most of these direct IL-1 inhibitory properties. Therefore, in addition to the previously reported antipyretic action of -MSH, this hormone can also inhibit two other known IL-1-sensitive cellular targets in vivo. Further, -MSH was incapable of modifying the comitogenic influence of IL-1 on murine thymocytes or on an IL-1 responsive T-cell line. These findings suggest a target cell specificity to the IL-1 inhibitory activities of -MSH and fail to support the hypothesis that -MSH functions through competitive inhibition of specific cellular receptors for IL-1.     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

Plasma endotoxin and cytokine levels in neutropenic and non-neutropenic bacteremic patients

Plasma endotoxin, tumor necrosis factor- (TNF-), interleukin 1 (IL-1), interleukin 1 receptor antagonist (IL-1ra), and interleukin 6 (IL-6) concentrations in 69 bacteremic patients were compared with those in 54 nonbacteremic patients suffering from suspected bacterial infections. Only three (11%) of the 27 patients with gram-negative bacteremia showed detectable levels of endotoxin. TNF- was detected in 6% of the bacteremic patients and in none of the nonbacteremic patients. Median IL-6 levels were significantly higher in bacteremic than in nonbacteremic patients (55 vs. 0 pg/ml, p=0.0008). IL-6 concentrations were similar in neutropenic and non-neutropenic bacteremic patients (median 55 vs. 74 pg/ml). In contrast, neutropenic bacteremic patients had significantly lower concentrations of tIL-1ra than non-neutropenic bacteremic patients (250 vs. 1,950 pg/ml, p<0.0001). Patients with fatal bacteremia had significantly higher concentrations of IL-6 and IL-1ra than the survivors (median, 450 vs. 40, p=0.012 and 7,600 vs. 420 pg/ml, p=0.0075, respectively). Determinations of endotoxin or TNF- in patients with suspected bacteremia failed to offer clinically relevant data on the prognosis of these patients. IL-6 levels correlated with both the presence of bacteremia and the risk of death. Granulocytopenic patients with bacteremia had lower levels of circulating IL-1ra than patients with normal granulocyte counts, and these levels correlated with poor outcome.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Interleukin 1β (IL-1β) release from fresh and cultured colonic mucosa in patients with ulcerative colitis (UC)

Interleukin-1, a cytokine produced by macrophages and other tissue cells, has a major role in inflammatory and immunological responses. Increased levels of IL-1 activity have been reported in experimental colitis and in patients with active Crohn's disease (CD) and ulcerative colitis (UC). IL-1 release from fresh and cultured colonic biopsies and IL-1 plasma concentrations was determined in 15 patients with active UC, 16 with UC in remission and 10 normal control subjects. Biopsies, taken at colonoscopy were weighed, washed in 1 ml of 0.9% sodium chloride solution and then cultured for 24 h in 10% fetal calf serum/RPMI. IL-1 activity was determined by ELISA KIT (Cystron Biotechnology) in plasma samples, washing solution and the incubation medium. Very low levels of IL-1 were detected only in 3 plasma samples, all from active patients. Significantly more IL-1 was released from fresh and cultured colonic mucosa obtained from patients with UC in remission compared to normal mucosa (p<0.01). Furthermore, specimens from active UC released significantly more IL-1 than those from patients in remission (p<0.01). In conclusion, IL-1 may play an important role in mediating the inflammatory response in UC.     

Effects of Heat Pretreatment on Histopathology, Cytokine Production, and Surfactant in Endotoxin-Induced Acute Lung Injury

To determine the effect of heat stress on histopathology of acute lung injury (ALI) caused by administration of lipopolysaccharide (LPS), and to determine the roles of tumor necrosis factor (TNF)-, interleukin (IL)-1, interferon (IFN)-, IL-10 and surfactants in heat-induced tolerance to ALI, we administered either saline or LPS (3 mg/kg of body weight) intravenously to male Sprague-Dawley rats without and with heat pretreatment. Five hours after LPS or saline treatment (23 h after heat-pretreatment), samples were obtained. We found that the histopathologic features of LPS-induced ALI were attenuated by heat-pretreatment. Heat-pretreatment did not decrease the elevated plasma or BAL fluid levels of TNF-, IL-1, and IFN- by LPS. The plasma level of IL-10 in LPS-treated rats with heat-pretreatment, however, was increased compared to that of LPS-treated rats without heat-pretreatment (P = 0.001). There were no differences in the BAL fluid concentrations of light or heavy density pulmonary surfactant phospholipids depending on heat-pretreatment in LPS-treated rats. These observations suggest that IL-10 might play a role in decreasing LPS-induced acute lung injury after heat-pretreatment.     

Role of interferon-γ on the in vivo expression of functional interleukin-2 receptors by murine macrophages

Interferon - activates both in vitro and in vivo macrophage functions. Injection of rat recombinant interferon- (rR-IFN-) induced the expression of interleukin-2 receptors (1L-2R) by peritoneal macrophages from normal BALB/c and MRL-+/+ mice. Moreover, rR-IFN- stimulated in a dose-dependent manner the oxidative burst of cells as revealed by luminol-dependent chemiluminescene (LDCL). Resident peritoneal macrophages from MRL-lpr/lpr (mice that develop a systemic lupus-like syndrome) showed a higher PMA-triggered LDCL response. This enhanced activity was accompanied by an increase in IL-2R expression (30% vs. < 1%). The activated; macrophages from rR-IFN--treated normal mice as well as MRL-lpr/lpr mice did not respond to the addition of recombinant interleukin-2 (rHu-IL-2) by an inc -ease in LDCL. However, rHu-IL-2 triggering became efficient when cells enriched in IL-2R-bearing macrophages were preincubated overnight with rHu-IL-2R. This response may point out a functional role for IL-2R and provide a role for IL-2 in certain macrophage functions.     

Nephritogenicity and α-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane

Nephritogenicity (anti-GBM-nephritis-inducing activity) and -chain composition of globular-domain (NC1) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different chains of type IV collagen. A purified nephritogenic fraction from renal BM contained 1–6(IV)NC1, whereas a non-nephritogenic fraction contained only 1–2(IV)NC1. Renal and pulmonary NC1 had strong nephritogenic activity; placental NC1 had weak activity. The renal and pulmonary fractions contained 1–6(IV)NC1, and the placental fraction had a large amount of 1–2(IV)NC1 and a very small amount of 3–6(IV)NC1. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained 1–5(IV) chains, but not the 6(IV) chain. The absence of 6(IV) chain in glomerular BM in bovine and in humans indicates that 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of 3–5(IV)NC1.     

Zur Polypeptidketten-Struktur von Immunglobulinen

Zusammenfassung Die Ergebnisse optischer Drehfähigkeitsmessungen an H- und L-Polypeptidketten normaler und Myelomproteine vom G-Typ werden mitgeteilt und mit den optischen Konstanten kompletter G-Proteine verglichen. Durch Kreuzungen zwischen unspezifischen und spezifischen Polypeptidketten werden die Bedingungen einer spezifischen Rekombination geprüft. Sie sind insbesonders dann gegeben, wenn autologe, d.h. vom gleichen Myelomprotein stammende Polypeptidketten gemischt werden. Die Befunde werden in ihrer Beziehung zum Problem der Antikörperspezifität diskutiert.

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Summary The results of measurements of the optical rotatory dispersion on H- and L-polypeptide chains of normal and myeloma proteins are reported Comparisons of the optical parameters with those of the whole G-proteins were performed. By mixing of normal or unspecific and myeloma or specific polypeptide chains the conditions of specific recombination were examined. Specific recombination occurs especially when autologous chains, that means polypeptide chains of an individual myeloma protein, are mixed. The implications of this finding are discussed with respect of the problem of antibody specifity.

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Herrn Prof. Dr.H. E. Bock zum 65. Geburtstag.     

The Pulmonary Inflammatory Response to Experimental Fecal Peritonitis: Relative Roles of Tumor Necrosis Factor-α and Endotoxin

The roles of endotoxin (LPS) and tumor necrosis factor- (TNF-) in the causation of organ injury during sepsis are unclear. To study LPS and TNF- in the genesis of lung inflammation after cecal ligation and puncture (CLP), we used endotoxin-resistant (C3H/HeJ) and endotoxin-sensitive mice (C3H/HeOuJ). We examined lung neutrophil sequestration, interleukin 1 (IL-1) mRNA expression, IL-1 protein expression, and injury. We also determined the expression of two C-X-C chemokine mRNAs, macrophage inflammatory protein-2 (MIP-2) and KC, in the lung to determine whether in vivo, endotoxin, or TNF- are significant modulators of MIP-2 and KC mRNA expression. After CLP, increased neutrophils sequestrated in the lungs of both strains of mice and coincided with an increase in expression of IL-1, MIP-2 and KC mRNAs, and IL-1 protein. Lung and serum TNF- were significantly increased in the C3H/HeOuJ strain but not in the C3H/HeJ strain. Histologic studies of the lung revealed similar injury in both strains. Our results suggest that bacterial factors other than endotoxin cause lung neutrophil sequestration and injury after CLP and further, that TNF- production is not a prerequisite. Our findings also suggest a potential role for local pulmonary chemokine production in the control of neutrophil sequestration after CLP.     

Isolation of the Bα and Bβ mating-type loci of Schizophyllum commune

The B mating type of the basidiomycete fungus, Schizophyllum commune is determined by two, tightly linked, multi-specificity (also called multi-allelic) loci: B and B. A plasmid library was used in DNA-mediated transformation to obtain transformants that displayed B-directed development. Plasmids that conferred B1 and B1 mating-type specificities were rescued from the transformants. Fragments of DNA from each plasmid hybridized to genomic DNA from the strain used to make the plasmid library; however, they did not hybridize, or hybridized only weakly, to genomic DNA from strains with mating-type specificities different from B1 or B1. The cloned fragments are presumed to correspond to active regions of each B mating-type locus.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.     

Zur Früherkennung der diabetischen Nephropathie: Beta2-Mikroglobulin im Serum

Summary Detection of early diabetic nephropathy is necessary to postpone or even prevent progression of irreversible kidney damage by therapeutic measures. Beta₂-microglobulin (₂-MG) as a parameter of the glomerular filtration rate has been measured by immunoassay in the serum of 100 diabetic subjects, 50 insulin-dependent (IDD), and 50 noninsulin-dependent (NIDD) patients. The results are compared with endogenous creatinine-clearance, serum creatinine concentration, and proteinuria and are related to different stages of diabetic retinopathy (RD). Normal values were obtained from 50 healthy age- and sex-matched subjects.A close correlation was found between ₂-MG levels and endogenous creatinine clearance. Thirty-nine diabetics revealed an elevated ₂-MG (2.5 mg/l or higher), only 16 of whom had increased serum creatinine levels (1.4 mg/dl or higher). Significant differences of ₂-MG were obtained between each group of patients with different stages of RD. A relevant difference of serum creatinine was found only between patients with normal eye fundus and advanced proliferative retinopathy, respectively. Without RD 26% of the patients revealed elevated ₂-MG but normal creatinine values demonstrating a latent nephropathy, just 8% showed an increase of both parameters. Of the diabetics with proliferative retinopathy 40% suffered from impaired kidney function proven by reduced creatinine clearance and by elevation of ₂-MG and creatinine as well, 15% just revealed an increase of ₂-MG with normal creatinine levels. The incidence and extent of nephropathy demonstrated by pathologic values of both ₂-MG and serum creatinine were significantly higher in IDD patients with a smaller proportion of latent nephropathy as compared to NIDD patients (p<0.02). This is also true for the markedly increased proteinuria in IDD subjects. In both groups, measurement of ₂-MG disclosed more often decrease of renal function in diabetics than did concentrations of serum creatinine.The determination of serum ₂-MG appears to be a reliable and sensitive method for the early detection of minor impairment of kidney function in diabetes mellitus.

Unterstützt durch die Deutsche Forschungsgemeinschaft (Go 299/2)     

Regulation of glycoprotein D synthesis of herpes simplex virus 1 by α 4 protein,the major regulatory protein of the virus,in stably transformed cell lines: effect of the relative gene copy numbers

Summary Earlier studies concerning 1 gene regulation by the 4 protein, the major regulatory protein of herpes simplex virus 1 (HSV-1), in stably transformed cell lines, reported conflicting results, i.e., 4 protein positively regulated the ₁ gB gene in 4/gB cells, while it negatively regulated the ₁ gD gene in 4/BJ cells. Both cell lines were derived from a common parental cell line 4/c 113 that contains 1 copy of the 4 gene, and the only apparent difference between them was the relative copy number of the gB and gD sequences (1 and 30–50, respectively) resident in the cell genome. We investigated this disparity by constructing a cell line (BA 4) that contains one copy each of the 4 and ₁ gD sequences, by fusion of 4/c 113 and BJt cells, containing and expressing respectively 1 copy of the 4 and gD genes. BA 4 cells constitutively expressed both the 4, gD genes inherited from the parental cell lines ( 4/c 113 and BJt). In BA 4 cells the 4 protein positively regulates the gD gene as evidenced from (i) higher levels of gD expression than the parental BJt cells lacking the 4 gene, and (ii) significant decrease in gD expression under conditions that render the 4 protein produced in BA 4 cells non-functional. In addition the ₂gG gene contained within the DNA fragment encoding the gD gene, is also expressed in BA 4 cells. On the basis of these data, we propose that gene regulation by the 4 protein is affected by the relative copy number of these genes, resident in the cell genome.     

Dose- and time-dependent effects of histamine on hypothalamic levels of interleukin-1β in rats

Some recent studies give support to the potential interaction between histamine (HA) and interleukin-1 (IL-1) in the central nervous system (CNS). At the peripheral level, HA acts as an immune modulator, but little is known on the neuroimmune role of this biogenic amine in the CNS. In the present study we have investigated the effects of HA, mepyramine, famotidine, thioperamide, and L-histidine on hypothalamic IL-1. HA induced a time- and dose-dependent decrease in the concentration of IL-1. The maximum effect was obtained 30 minutes after injection. The HA-induced IL-1 response in the hypothalamus was not inhibited by either mepyramine, famotidine or thioperamide. In addition, L-histidine exerted the same effect as HA. The interaction between HA and IL-1 in the CNS might be linked to neuroendocrine regulation as well as to neurotrophic activity and neuroimmune function.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.