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PubMed1982

Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes.

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab')(2) anti-CR(1) (C3b receptor) or F(ab')(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab')(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab')(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab' anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant.

Cell Membrane↗

PubMed1995

Kinetic modeling and mathematical analysis indicate that acute phase gene expression in Hep 3B cells is regulated by both transcriptional and posttranscriptional mechanisms.

To evaluate the possible role of posttranscriptional mechanisms in the acute phase response, we determined the kinetics of transcription (by nuclear run-on assay) and mRNA accumulation of five human acute phase genes in Hep 3B cells incubated with conditioned medium from LPS-stimulated monocytes. Increase in mRNA accumulation was comparable to increase in transcription rate for fibrinogen-alpha and alpha-1 protease inhibitor, suggesting largely transcriptional regulation. In contrast, mRNA accumulation was about 10-20-fold greater than transcriptional increase for serum amyloid A, C3, and factor B, suggesting participation of posttranscriptional mechanisms. Since finding a disparity between the magnitudes of increase in mRNA and transcription does not definitively establish involvement of posttranscriptional mechanisms, we subjected our data to modeling studies and dynamic mathematical analysis to evaluate this possibility more rigorously. In modeling studies, accumulation curves resembling those observed for these three mRNAs could be generated from the nuclear run-on results only if posttranscriptional regulation was assumed. Dynamic mathematical analysis of relative transcription rates and relative mRNA abundance also strongly supported participation of posttranscriptional mechanisms. These observations suggest that posttranscriptional regulation plays a substantial role in induction of some, but not all acute phase proteins.

Acute-Phase Reaction↗

PubMed1978

Release of granulocyte elastase in lethal canine endotoxin shock.

The release of granulocyte elastase and its interaction with plasma protease inhibitors was studied in dogs receiving a slow infusion of a lethal dose of Escherichia coli endotoxin. During endotoxin infusion a marked decline in leucocyte counts was parallelled by a rapid increase in plasma granulocyte elastase concentrations. Maximal values were reached after 3 h, when the infusion was ended. Crossed immunoelectrophoresis with antiserum against granulocyte elastase did not reveal the presence of elastase components with the electrophoretic mobility of free elastase, but elastase-alpha1-antitrypsin complexes were detected. A gradually decreasing plasma concentration of alpha2-macroglobulin was noted during the experiments. Crossed immunoelectrophoresis, however, did not reveal any electrophoretic heterogeneity. It is concluded that the release of granulocyte proteases might be of significance for several pathophysiological changes seen in endotoxin shock.

Animals↗

PubMed1998

Interactions between Candida species and platelets.

Candida spp. are able to cause disseminated disease in immunocompromised patients. This study examined the interactions of Candida spp. with platelets, complement and polymorphonuclear leucocytes (PMNLs). With the exception of C. albicans, all other Candida spp., including a C. albicans strain previously classified as C. stellatoidia, aggregated human platelets at a ratio of yeast cells: platelets of 1:80. Usually, those species and strains that aggregated platelets were either killed or prevented from growing in platelet-rich plasma indicating that the aggregation released microbicidal or microbistatic substances that were active against Candida spp. All Candida spp. were resistant to attack by complement in 50% serum. However, all species activated complement as determined by the presence of C3 fragments on their surface, in particular a 195-kDa fragment corresponding to C3c, two fragments at 67 and 40 kDa corresponding to iC3b, and a 33-kDa fragment corresponding to C3d. When strains were tested for their ability to stimulate the release of pro-inflammatory substances from platelets and PMNLs, it was found that most strains stimulated PMNLs to release interleukin(IL)-8 but not IL-1beta or leucotriene B4. The ability of C. albicans to evade complement-mediated killing and not to aggregate platelets may contribute to the survival of this species in the blood during vascular infections.

Anticoagulants↗

PubMed1996

Complement activation on thiol-modified gold surfaces.

To evaluate the importance of protein adsorption and chemical composition of the solid surface on complement activation, we used mercaptoglycerol (MG) and mercaptopropionic acid (MPA) modified gold surfaces as model surfaces. The complement activation by these surfaces was determined by measuring fluid phase iC3b and C5b-9 in serum that had been in contact with the surfaces. In addition, "active" C3 deposition at the modified surfaces was measured with the use of ellipsometry, an optical technique. It was found that the MG surface caused pronounced productions of iC3b and C5b-9 in serum as well as increased C3 deposition on the surface. In contrast, the bare gold surface and the MPA surface caused very little complement activation. The MG surface seems to have a high affinity with immunoglobulin G (IgG) that may be one explanation for the high complement activation ability of the MG surface. However, complement activation at the MG surface was relatively insensitive to Mg-EGTA buffer at a high serum concentration, indicating alternative pathway activation when classical pathway activation was inhibited. Corresponding control experiments performed with Mg-EGTA serum at hydrophobic silicon surfaces precoated with IgG showed no sign of alternative pathway activation. At a lower serum concentration, classical pathway activation seemed to dominate at the MG surface and the hydrophobic silicon surface precoated with IgG. Most probably, it is the hydroxyl groups at the MG surface that are associated with alternative pathway activation.

3-Mercaptopropionic Acid↗

PubMed1999

Complement activation accelerates glomerular injury in diabetic rats.

A known inhibitor of the complement cascade (K-76 COONa) was administered to an inbred diabetic rat model to investigate whether the complement system may play a role in the progression of diabetic glomerulosclerosis. Drinking water containing K-76 COONa was available continuously to inbred diabetic rats (Otsuka Long-Evans Tokushima Fatty, OLETF) from the age of 25 to 55 weeks (Group L). Drinking water without K-76 COONa was similarly available to OLETF rats (Group H) and nondiabetic control rats (Long-Evans Tokushima Otsuka, LETO) (Group C). The levels of plasma glucose (mg/dl) at the 55th week were 156 +/- 16 in Group C, 252 +/- 18 in Group L and 349 +/- 93 in Group H. There was no significant difference in the degree of diabetes between Group L and Group H. The levels of urinary protein at 55 weeks of age (mg/day) were 2.1 +/- 0.4 in Group C, 11. 6 +/- 1.5 in Group L and 18.0 +/- 2.8 in Group H. The level of urinary protein was significantly decreased by the administration of K-76 COONa. Histological examination of renal specimens from the sacrificed rats at 55 weeks of age revealed diffuse mesangial expansion in almost all glomeruli in Group H, exudative lesions in 30% of glomeruli in Group H, and only mild mesangial expansion was recognized in Group L. Immunofluorescence study revealed brilliant staining of C3 and immunoglobulins (Ig) in Group H; trace staining of Ig and no staining of C3 were recognized in Group L. The incubation study with guinea pig serum and glomeruli from rats revealed that Ig and complement components also bound to injured glomeruli in vitro. These data indicate that the complement cascade is activated by injured glomeruli and this activation exacerbates diabetic glomerulosclerosis.

Animals↗

PubMed1990

C4a anaphylatoxin levels as an indicator of disease activity in systemic lupus erythematosus.

The use of a synthetic protease inhibitor, nafamstat mesilate, has enabled reliable estimations of in vivo complement activation to be made in systemic lupus erythematosus (SLE). Elevation of C3a anaphylatoxins was found in two out of 24 patients and elevation of C4a anaphylatoxins was found in 20 out of 24 patients, confirming that complement activation, predominantly by the classical pathway, is a common occurrence in the disease. Significantly higher levels of C4a anaphylatoxin were found in 16 patients, with more aggressive disease requiring supplementary treatment with azathioprine, while the remaining eight patients, with less severe disease, required purely steroid therapy. Very strong associations between elevated C4a anaphylatoxins and raised DNA antibody titres, C1q binding activity and low complement C4 levels were also observed, suggesting that anaphylatoxin measurement may be a sensitive additional method for monitoring disease activity in SLE.

Adult↗

PubMed2006

Interaction of mannan binding lectin with alpha2 macroglobulin via exposed oligomannose glycans: a conserved feature of the thiol ester protein family?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.

Animals↗

PubMed1988

[Comparison of protein concentrations in saliva and serum].

Saliva and serum protein profiles composed of 12 proteins: IgA, IgG, IgM, albumin, transferrin, alpha 1-glycoprotein acid, alpha 2-macroglobulin, alpha 1-proteinase inhibitor, haptoglobine, C3C, C4 and CRP were obtained by laser nephelometry of aliquots of samples of non-stimulated whole saliva and blood taken at the same time. The population of 138 controls of both sexes was divided into 4 age groups: 1 (20 to 29 years), 2 (30 to 39 years), 3 (40 to 49 years), 4 (50 to 60 years). Using the techniques developed for saliva assay, 10 proteins were measurable: IgA, IgG, IgM, albumin, transferrin, alpha 1-glycoprotein acid, alpha 2-macroglobulin, alpha 1-proteinase inhibitor, haptoglobulin and C3C. The C4 fraction and CRP were present at concentrations lower than the detection limit i.e. at 1.8 and 2.05 mg/l. In the serum 11 proteins were analysed, CRP occurring at less than 0.01 g/l. The statistical two-factor variance analysis test (sex and age) demonstrated the statistically significant influence of age in the saliva for alpha 1-PI and alpha 2-M in the serum for Tf, alpha 2-M and alpha 1-PI, and sex in the serum for IgG, IgM, alpha 1-PI, alpha 2-M and also the interaction of both factors in the serum for alpha 2-M.

Adult↗

PubMed1990

Regulation of the hepatic synthesis of C1 inhibitor by the hepatocyte stimulating factors interleukin 6 and interferon gamma.

C1 inhibitor (C1INH), the major plasma inhibitor of activated C1, kallikrein, and activated Hageman factor, may be an important factor in limiting inflammatory injury mediated by the complement and contact systems. C1INH is thought to be synthesized primarily in the liver; however, the regulators of hepatic C1 inhibitor synthesis are completely unknown. In this report, we analyze the regulation of C1INH synthesis by hepatocyte stimulating factors in human hepatoma cell lines and primary hepatocytes. Interleukin-6 and interferon gamma increase C1INH production in both hepatoma cells and hepatocytes. These cytokines stimulate de novo synthesis of functional C1INH, acting at a pretranslational level as assessed by Northern blotting. The stimulatory effects of interleukin-6 and interferon gamma on C1INH synthesis are separate and are differentially modulated by interleukin-1. These results establish that hepatic C1INH synthesis is regulated by hepatocyte stimulating factors and reveal novel interactions between these factors.

Acute-Phase Proteins↗

PubMed2003

Human C1 esterase inhibitor attenuates murine mesenteric ischemia/reperfusion induced local organ injury.

BACKGROUND: Complement activation contributes to ischemia and reperfusion (IR)-initiated organ injury. C1 inhibitor (C1 Inh) inhibits the earliest steps of the classical and the mannose binding lectin pathways. MATERIALS AND METHODS: To determine whether C1 Inh prevented tissue injury, we performed intestinal IR experiments in BALB/c and C57BL/6 mice. RESULTS: We found that C1 Inh limits mucosal injury in the two strains in a dose dependent manner. Tissue damage was associated with the accumulation of functional polymorphonuclear cells, which was reduced following C1 Inh treatment. Constitutive nitric oxide synthase activity correlated with the development of injury in the C57BL/6 but not in the BALB/c mouse. CONCLUSIONS: These findings emphasize the importance of complement activation in ischemia/reperfusion and highlight the potential therapeutic use of C1 Inh in limiting or preventing damage caused by IR.

Animals↗

PubMed1975

Immune reactions in acute viral hepatitis.

Serial studies of PHA-induced lymphocyte transformation, serum autoantibodies, immunoglobulins and complement were performed in seventeen patients with hepatitis A and nine patients with hepatitis B. In both types of hepatitis PHA-induced transformation was markedly impaired during the 1st week after the onset of jaundice and there was less marked but prolonged impairment for a further period of 6-10 weeks. A group of eleven subjects with a previous history of hepatitis had values which were similar to those of healthy persons. Serum from patients with hepatitis A and hepatitis B contains an inhibitor of lymphocyte response to PHA. The inhibitor depresses the function of both patients' and normal lymphocytes and is only detectable during the acute phase of the illness. Washing lymphocytes free from autologous serum did not restore the PHA response to normal but the markedly impaired response present during the first 2 weeks of the illness was improved. A serum factor or factors may therefore be responsible for at least part of the impaired response of lymphocytes to PHA during the acute phase of hepatitis but does not appear to account for the more prolonged impairment of the PHA response. The protracted lymphocyte defect is possibly induced by hepatitis virus. The incidence of autoantibodies and the changes in immunoglobulin levels were similar to those reported by other workers.

Antibody Formation↗

PubMed1999

IgA nephropathy. From molecules to men.

The author of this volume has studied IgA nephropathy for nearly 25 years, almost as long as primary IgA nephropathy has been recognized as a new disease. IgA nephropathy, considered to be an immune-complex-mediated glomerulonephritis, is characterized by granular deposition of IgA (mainly IgA1) and C3 in the glomerular mesangial areas and is defined as nephropathy showing proliferative changes in the glomerular mesangial cells and increases in the mesangial matrices. Apart from being one of the most common types of chronic glomerulonephritis, it is also the most frequent case of end-stage renal disease. Since the pathogenesis of IgA nephropathy is still obscure, specific treatment is not yet available. Previous approaches have included tonsillectomy, anticoagulants, prednisolone, immunosuppressants, angiotensin-converting enzyme inhibitors and others. During his career, the author of this book has studied many aspects of IgA nephropathy, shedding much light on the mechanism of development and progression of this disease. He also undertook new treatments for patients and developed animal models for IgA nephropathy. The purpose of the present volume is to review the author's work on pathogenesis and treatment of the disease and to provide the most up-to-date findings on this subject, constituting a valuable source of information for nephrologists, general practitioners, residents and interns.

Animals↗

PubMed1985

On the kinetics of complement activation, leucopenia and granulocyte-elastase release induced by haemodialysis.

In order to elucidate the kinetics of haemodialysis-induced activation of complement, leucopenia and release of granulocyte-elastase, 10 patients (three females and seven males; mean age 47.8 years) were extensively studied during a 4 h haemodialysis treatment and for the following 24 h, and further compared with a healthy control group. Prior to dialysis patients had normal leucocyte count, plasma elastase bound to alpha 1-proteinase inhibitor (E-alpha 1P1) and total haemolytic complement, whereas plasma C3d was higher and plasma C5a lower than in controls. Haemodialysis induced initial leucopenia and subsequent rebound phenomenon lasting 24 h post treatment. These alterations were due to almost selective changes in neutrophile count as monocyte and lymphocyte counts, apart from decrease in the first 30 min, were unchanged. Total haemolytic complement decreased initially during dialysis and rose at the end. Generation of C5a within the dialyser was evident by demonstration of high levels of this anaphylatoxin in dialyser effluent plasma; maximal values observed coincided with the nadir of leukopenia. Plasma C3d and E-alpha 1P1 both progressively rose during dialysis. After termination of extracorporeal circulation the disappearance rates (T/2) were approximately 6 h and 2.5 h respectively. Haemodialysis thus induces changes in the complement and leucocyte system resembling an acute inflammation, which out-lasts the treatment period.

Adult↗

PubMed2003

Decreased ratio of CD4/CD8 lymphocytes might be predictive for successful interferon alpha and lamivudine combined therapy in childhood chronic hepatitis B infection: A preliminary study.

BACKGROUND: In the development of chronic hepatitis with hepatitis B virus infection and in response to therapy, the immune status of the infected host plays a critical role. In this study, immunological variables were assessed in patients before interferon alpha and lamivudine therapy to determine if any pretreatment immunological parameter could be an indicator of response to therapy in childhood chronic hepatitis B infection. METHODS: Forty-four patients with chronic hepatitis B virus infection, aged 9.0 +/- 3.9 years, were enrolled in the study. The pretreatment clinical features, biochemical test results, histological activity indexes and immunological parameters were evaluated. All patients received interferon alpha for 6 months and lamivudine for 52 weeks. Four patients who could not be followed up were excluded from the study. The other 40 patients have been followed for a mean period of 27.5 +/- 9.7 months after therapy discontinuation. RESULTS: Seventeen patients showed loss of hepatitis B early antigen (HBeAg) with appearance of anti-HBe (42.5%) and six of those who responded also showed loss of hepatitis B surface antigen (HBsAg) with the presence of anti-HBs (15%). Except elevated aspartate aminotransferase levels, there was no significantly correlation between response and sex, age, pretreatment duration of disease and histological activity indexes. Pretreatment immunoglobulins (Ig), IgG subclasses, complement C3, C4 and secretory IgA levels were also not found to be significantly related to response. The evaluation of lymphocyte subsets showed that therapy responders had significantly reduced pretreatment ratios of CD4/CD8+ lymphocytes due to prominent increased percentages of CD8+ cells. The other cellular immunity parameters and some cell surface adhesion molecules were similar in both groups. CONCLUSION: This study emphasizes the importance of increased pretreatment CD8+ lymphocyte percentages leading to a significant decrease in CD4/CD8 ratio in chronic hepatitis B virus infection of childhood as an immunological factor predicting response to treatment.

Adolescent↗

PubMed1991

Rapid increases in the membrane expression of neutral endopeptidase (CD10), aminopeptidase N (CD13), tyrosine phosphatase (CD45), and Fc gamma-RIII (CD16) upon stimulation of human peripheral leukocytes with human C5a.

Rapid increases in the membrane expression of C3 receptors on granulocytes and monocytes in response to the anaphylatoxin C5a have previously been described. In this study we demonstrate increases in the membrane expression of neutral endopeptidase (NEP, CD10, CALLA), aminopeptidase N (APN, CD13), tyrosine phosphatase (CD45/CD45Ro) and the Fc R Fc gamma-RIII (CD16) on granulocytes within minutes of treatment with human C5a. Monocytes responded to C5a with increases in CD13 and CD45/CD45Ro. These membrane modulations could be prevented by preincubating the C5a preparations with anti-C5a mAb C17/5 but not by pretreating the cells with cycloheximide. Increases of CD10, CD13, and CD11b but not CD11a (LFA-1) were also observed in leukocytes from patients undergoing hemodialysis with cuprophan membranes. The increase of CD16 on granulocytes was dependent on the presence of plasma during in vitro activation with C5a indicating that plasma contains inhibitors which prevent the previously described loss of Fc gamma-RIII upon stimulation of the cells.

Aminopeptidases↗

PubMed1986

[Value of biochemical and imaging procedures for the diagnosis and prognosis of acute pancreatitis--results of a prospective clinical study].

For a period of 14 days we carried out measurements for alpha-1-protease inhibitor, alpha-2-macroglobulin, complement C 3, complement C 4, and C-reactive protein in two different groups of patients with acute pancreatitis. Group I consisted of 13 patients with edematous-interstitial pancreatitis and group II of 22 patients with necrotizing pancreatitis. Diagnosis of acute pancreatitis was established by clinical signs and symptoms, by specific pancreatic enzymes determined in the serum, by imaging procedures, and by laparotomy in 24 cases. The overall detection rate for pancreatic necrosis was 90% for the contrast enhanced CT and 33% for ultrasonography respectively. There were significant differences as to all measured serum parameters between the two morphologically defined pancreatitis groups. The necrosis detection rate was 95% for CRP and 85% for alpha-2-macroglobulin. The combined determination of CRP and alpha-2-macroglobulin is recommended in patients with acute pancreatitis to stage the severity of the disease and to probably replace the CT investigation.

Acute Disease↗

PubMed1991

Wheat germ agglutinin and other selected lectins increase synthesis of decay-accelerating factor in human endothelial cells.

Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N-acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N-acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N-acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium.

Antigens, CD↗