Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells.

The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn2+-dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+-dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+-dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.

Assessment of autoimmunity in patients with chronic urticaria.

BACKGROUND: The etiology of chronic urticaria is unknown, and an exogenous allergen cannot be identified as the cause in the vast majority of subjects. Thus the concept has evolved that it might be autoimmune. OBJECTIVE: We have prospectively assessed sera obtained from 50 consecutive patients with chronic urticaria for the presence of autoantibodies that could be pathogenic. METHODS: We tested sera for their ability to release histamine from human basophils and to activate rat basophil leukemia cells that were transfected with the alpha subunit of the IgE receptor. We also tested selected sera for anti-IgE antibodies and for IgG anti-Fc epsilon RI alpha by Western blot. RESULTS: Sera from 38 of 50 patients with chronic urticaria released beta-hexosaminidase from transfected rat basophil leukemia cells, whereas only one of 20 control sera did so (p < 0.001); in 30 subjects this could be attributed to IgG anti- Fc epsilon RI alpha. When human basophils were used, sera from 20 of 50 patients with chronic urticaria released a significant quantity of histamine compared with one of 20 control subjects (p < 0.01). Six patients with chronic urticaria and one control subject had IgG anti-IgE. In selected sera we could demonstrate IgG anti-Fc epsilon RI alpha by Western blot; however, some sera are positive for histamine release but do not demonstrate such binding. CONCLUSION: A large fraction of patients with chronic urticaria have antibody directed to Fc epsilon RI alpha that is functional (60%). A smaller number have IgG anti-IgE (10%). A third group may also have circulating factors capable of activating basophils or mast cells of which the identity is unknown. Thus chronic urticaria may be autoimmune in origin.

Assessment of kininases in rheumatic diseases and the effect of therapeutic agents.

Bradykinin is degraded in human plasma by a carboxypeptidase to yield desArg9-bradykinin (DBK) which is then digested by angiotensin-converting enzyme (ACE) to the pentapeptide Arg-Pro-Pro-Gly-Phe and the tripeptide Ser-Pro-Phe. We have studied the rate of kinin degradation by each of these enzymes in patients with rheumatoid arthritis (RA) and with systemic lupus erythematosus (SLE), compared with the degradation rate in degenerative joint disease and normal subjects. Carboxypeptidase activity was the same in all individuals, but ACE activity was increased in the RA and SLE patients. We examined the effects of aspirin, sodium salicylate, auranofin, penicillamine, and corticosteroids on kinin metabolism, and all of these were marked inhibitors of ACE; however, only penicillamine had any demonstrable inhibition of carboxypeptidase. These observations suggest rapid degradation of DBK in patients with untreated RA and SLE, whereas drugs utilized in therapy have the opposite effects. Studies to examine the role of DBK in disease manifestations are in progress.

Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme-linked differential antibody immunosorbent assay.

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF-deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF-deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time-dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.

Assessment of histamine release and kinin formation in man: identification of kinin degradation products and characterization of a lymphocyte-dependent histamine releasing factor.

Factors traditionally associated with histamine release include IgE antibody plus antigen and the anaphylatoxins C3a, C4a, and C5a. Yet histamine release is thought to occur in disorders such as chronic urticaria, atopic dermatitis, and contact dermatitis in which the above mechanisms do not appear operative. We have partially purified a factor from stimulated human mononuclear cells which causes basophil histamine release. It is homogeneous by gel filtration with a molecular weight of about 35,000 daltons and has two molecular forms when assessed by ion exchange chromatography, isoelectrofocusing in gels and chromatofocusing. The purified material, when radiolabeled, gives a single band upon two-dimensional gel electrophoresis and radioautography. This factor may therefore represent one mechanism in which delayed hypersensitivity and histamine release are linked. We are also developing methods to better assess the kinin-forming system in allergic diseases. Assays for enzyme inhibitor complexes are the most sensitive and specific methods for inferring activation in plasma. These include quantitation of activated Hageman factor-C1 INH complexes and kallikrein-C1 INH complexes each of which appears elevated in cutaneous late-phase reactions. However, bradykinin assessment is fraught with difficulties including spurious generation and rapid inactivation. Using high performance liquid chromatography we have separated bradykinin from kallidin, des-Arg9-bradykinin (the degradation product of carboxypeptidase N) as well as the fragments Arg-Pro-Pro-Gly-Phe, Ser-Pro, and Phe-Arg, the degradation products formed by angiotensin-converting enzyme. These can be assayed in purified mixtures, can be detected upon addition of bradykinin to human plasma and are formed by kaolin treatment of plasma.