Activation and function of human Hageman factor. The role of high molecular weight kininogen and prekallikrein.

The activation and function of surface-bound Hageman factor in human plasma are dependent upon both high molecular weight (HMW) kininogen and prekallikrein. HMW kininogen does not affect the binding of Hageman factor to surfaces, but it enhances the function of surface-bound Hageman factor as assessed by its ability to activate prekallikrein and Factor XI. The initial conversion of prekallikrein to kallikrein by the surface-bound Hageman factor in the presence of HMW kininogen is followed by a rapid enzymatic activation of Hageman factor by kallikrein. The latter interaction is also facilitated by HMW kininogen. Kallikrein therefore functions as an activator of Hageman factor by a positive feedback mechanism and generates most of the activated Hageman factor during brief exposure of plasma to activating surfaces. HMW kininogen is a cofactor in the enzymatic activation of Hageman factor by kallikrein and it also augments the function of the activated Hageman factor generated. The stoichiometry of the Hagman factor interaction with HMW kininogen suggests that it enhances the activity of the active site of Hageman factor. Since HMW kininogen and prekallikrein circulate as a complex, HMW kininogen may also place the prekallikrein in an optimal position for its reciprocal interaction with Hageman factor to proceed. The surface appears to play a passive role upon which bound Hageman factor and the prekallikrein-HMW kininogen complex can interact.

Anaphylactic release of a prekallikrein activator from human lung in vitro.

We have demonstrated the in vitro IgE-mediated release of a prekallikrein activator from human lung. The lung prekallikrein activator was partially purified by sequential chromatography on sulfopropyl-Sephadex, DEAE-Sephacel, and Sepharose 6B. Purified human prekallikrein was converted to its active form (kallikrein) by the lung protease. The generated kallikrein was shown to be biologically active; that is, it generates bradykinin from purified human high-molecular weight kininogen and also cleaves benzoyl-propyl-phenyl-arginyl-p-nitroanilide, a known synthetic substrate of kallikrein. The lung prekallikrein activator differs from the known physiologic activators of prekallikrein (the activated forms of Hageman factor) with respect to: (a) size (it has a mol wt of approximately 175,000); (b) synthetic substrate specificity (D-propyl/phenyl/arginyl-p-nitroanilide is a substrate for the activated forms of Hageman factor, but not the lung protease); (c) antigenic specificity (an anti-Hageman factor immunoadsorbent column did not remove significant amounts of the lung protease, while it removed most of the activity of activated Hageman factor fragments); and (d) inhibition profile (the lung proteases was not inhibited by corn trypsin inhibitor). This prekallikrein activator provides a physiologic mechanism by which prekallikrein can be directly activated during IgE-mediated reactions of the lung. While the role of this lung prekallikrein activator in immediate hypersensitivity reactions and in other inflammatory processes is not clear, it does represent a first and important interface between IgE-mediated reactions and the Hageman factor-dependent pathways of the inflammatory response.

Nasal challenge with ragweed pollen in hay fever patients. Effect of immunotherapy.

Challenge of the nasal mucosa of allergic subjects with specific allergen induces not only the expected sneezing and rhinorrhea, but also the appearance in nasal secretions of mediators commonly associated with activation of mast cells or basophils: histamine, leukotrienes, prostaglandin D2 (PGD2), kinins, and TAME ([3H]-N-alpha-tosyl-L-arginine methyl ester)-esterase. To determine whether specific immunotherapy alters mediator release in vivo, nasal pollen challenge was used to compare 27 untreated highly sensitive ragweed (RW)-allergic subjects with 12 similarly sensitive patients receiving long-term immunotherapy (3-5 yr) with RW extract (median dose, 6 micrograms RW antigen E). The two groups were equally sensitive based on skin tests and basophil histamine release. The immunized group had a diminished response as demonstrated by (a) the treated group required higher pollen doses to excite sneezing or mediator release; (b) significantly fewer subjects in the treated group released mediators at any dose (TAME-esterase [P = 0.005], PGD2 [P = 0.04]), and (c) the treated group released 3-5-fold less mediator (TAME-esterase [P = 0.01], and histamine [P = 0.02]).

Alterations in human lymphocyte DNA caused by sulfur mustard can be mitigated by selective inhibitors of poly(ADP-ribose) polymerase.

Changes in genomic DNA caused by exposure to the cytotoxic alkylating agent, 2,2'-dichlorodiethyl sulfide (sulfur mustard; HD), alone or in combination with selective inhibitors of poly(ADP-ribose) polymerase (PARP), were analyzed as a function of HD concentration and post-exposure time. Preparations of human peripheral blood lymphocytes were exposed to HD (1x10(-8) M-1x10(-3) M), and incubated at 37 degrees C for 0-24 h. Total genomic DNA was extracted from these cells and compared with DNA from control cells of the same donor using agarose gel electrophoresis. The effects of HD on genomic DNA depended on the HD concentration and the length of the post-exposure time interval. DNA fragmentation was detected as early as 2 h after exposure to 3x10(-4) M HD, or at 24 h after exposure to 6x10(-6) M HD. The qualitative DNA pattern, as well as the extent of DNA fragmentation, changed with post-exposure time. Exposure to HD caused a time-dependent shift in the DNA cleavage pattern from an oligonucleosome-sized 'DNA ladder' characteristic of apoptotic cell death, to a 'broad band' pattern characteristic of necrotic cell death. DNA fragmentation was not observed if cells were killed with heat or with Lewisite. Treatment of cells with selective PARP inhibitors consistently altered the DNA fragmentation caused by HD exposure. The inhibitors arrested DNA fragmentation at the DNA ladder stage. This effect only was observed if the PARP inhibitors were applied within 8 h of HD exposure. We conclude that early inhibition of PARP activity can induce a switch in the mechanism of cell death caused by HD. Such a switch may be useful therapeutically to convert a lytic, pro-inflammatory cell death that includes the disintegration of dying cells (necrosis), into a slower, programmed cell death that includes absorption of dying cells (apoptosis).

Exposure of human lymphocytes to bis-(2-chloroethyl)sulfide solubilizes truncated and intact core histones.

Bis-(2-chloroethyl)sulfide (BCES) is a radiomimetic, bifunctional alkylating agent that cross-links DNA, disrupts higher-order nuclear structure and selectively kills rapidly proliferating cell types. While chemically fractionating primary, human lymphocytes after challenge with cytotoxic doses of BCES, we detected a 12,900 M(r) polypeptide in 1.0 M NaCl extracts of exposed cells that was markedly increased compared to controls. By computer-aided image analysis of polyacrylamide gels, it was detected as early as 4 h following 1 mM BCES and increased approximately 10-fold by 24 h. Two other polypeptides of 16,320 and 16,970 M(r) also were increased measurably at 24 h following BCES exposure. Altered polypeptides were found in 28 of 28 separate lymphocyte preparations ranging in cell density from 5 x 10(6)/ml to 6 x 10(7)/ml. They were not present if cells were killed with equimolar concentrations of a different cytotoxic agent, chlorovinyl-dichloroarsine (lewisite). Appearance of the polypeptides was unaffected by sulfhydryl reducing agents or pretreatment of cells with the protein synthesis inhibitor, cycloheximide. Micro sequencing resulted in a perfect match of the 12,900 M(r) polypeptide amino terminus with residues 19-27 of histone H2B. This corresponds to the exact site of H2B cleavage obtained when intact nucleosomes are treated with chymotrypsin. Sequence data from the other two altered polypeptides identified them as intact histone H2B and histone H3. Lymphocyte genomic DNA integrity also was assessed after BCES exposure and found to undergo extensive fragmentation typical of cellular necrosis. We speculate that exposure of isolated cells to BCES disrupts nucleosome structure by mechanism(s) that involve abnormal removal and perhaps proteolysis of core histones.

Basophil mediators and their release, with emphasis on BK-A.

During IgE-mediated events, basophils secrete small molecular weight mediators (histamine, slow reacting substance, Eosinophil chemotactic factor) which are thought to participate in inflammatory processes. We here describe the IgE-mediated secretion of large molecular weight mediators which have the potential for the activation of the Hageman factor dependent pathways, and the generation of biologically active peptides. These large molecular weight basophil derived mediators may, through the activation of the Hageman factor dependent pathways, influence mechanisms which participate in both acute and chronic cell-mediated inflammatory processes. We suggest that these proteases may participate not only as mediators of the immediate hypersensitivity reaction, but may also function in important aspects of the entire inflammatory response.

Regulation by serine esterase of histamine release from human leukocytes--I. Direct release of histamine by the serine esterase inhibitors diisopropyl fluorophosphate (DFP) and soman (GD).

The serine esterase inhibitor diisopropyl fluorophosphate (DFP) had been reported previously to inhibit IgE-dependent histamine release. Recently, it has been demonstrated that lower concentrations of DFP enhance IgE-dependent histamine release and inhibit desensitization. This manuscript describes the abilities of several esterase inhibitors to cause release of histamine from human leukocytes (basophils), by a process that is IgE-independent. This esterase inhibitor-induced histamine release appears to be by a non-cytotoxic mechanism that requires calcium and is temperature dependent. These histamine release processes occurred over a longer period of time than IgE-dependent release. Direct release of histamine by these small molecular weight inhibitors and inhibition of desensitization both suggest that one or more serine esterases are involved in the regulation of histamine release from human basophils.

2,2'-Dichlorodiethyl sulfide (sulfur mustard) decreases NAD+ levels in human leukocytes.

2,2'-Dichlorodiethyl sulfide (sulfur mustard, HD) extensively alkylates DNA in a concentration-dependent manner in many cell types. We have proposed a biochemical hypothesis that explains HD-induced injury by linking DNA alkylation and DNA breaks with activation of poly(ADP-ribose) polymerase, resulting in depletion of cellular NAD+. This hypothesis was tested by treating human leukocytes with HD to determine whether NAD+ depletion occurred as predicted. These cells demonstrated a decrease in NAD+ levels which was dependent on both concentration of HD and time after exposure. Inhibitors of poly(ADP-ribose) polymerase or substrates for NAD+ synthesis were able to prevent the HD-induced NAD+ decrease.

Release of elastase from purified human lung mast cells and basophils. Identification as a Hageman factor cleaving enzyme.

Elastase, a serine protease, is capable of inducing severe lung destruction in experimental animal models. We now report that this proteinase exists preformed in neutrophil-free sonicates of purified human lung mast cells (greater than 98% purity) and in circulating peripheral blood basophils (greater than 97% purity). The elastase levels in both cell types (41-174 ng/10(6) cells) represents approximately 3-20% of those found in human neutrophils; both cell types released their elastase following anti-IgE and ionophore A23187 challenge. The apparent molecular size of the mast cell enzyme on Sephadex G-100 gel filtration, as well as its inhibition profile, was identical to that of purified human neutrophil elastase. This mast cell elastase is identical to our previously reported mast cell-derived Hageman factor cleaving activity. Mast cell-, basophil-, and neutrophil-derived elastases cleave Hageman factor into fragments of 52,000 and 28,000 Da; cleavage by all three enzymes is inhibited by preincubation with polyclonal antibodies directed against human neutrophil elastase.

The time-dependent effect of 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD, 1,1'-thiobis [2-chloroethane]) on the lymphocyte viability and the kinetics of protection by poly(ADP-ribose) polymerase inhibitors.

2,2'-Dichlorodiethyl sulfide (sulfur mustard, HD, 1,1'-thiobis [2-chloroethane]) is a potent vesicant which can cause severe lesions to skin, lung, and eyes. Due to the high number of debilitating exposures during the Iran-Iraq war to the alkylating agent, HD, there is an increased interest in its mechanism of action and in the development of therapeutic interventions to prevent HD-induced lesions. Recently we reported an in vitro assay using human mononuclear leukocytes for studying HD-induced pathology. To study the time dependence of HD-induced mononuclear leukocyte cell death and to determine the parameters of any potential therapeutic intervention, an assay was developed and automated using a flow cytometer to measure propidium iodide exclusion by mononuclear cells. This assay demonstrated that HD-initiated cell death did not begin before 4 h post-exposure, but after 4 h proceeded in a concentration-dependent manner. In this assay, both niacinamide and 3-aminobenzamide, poly(ADP-ribose) polymerase inhibitors, were shown to be effective in blocking HD-induced cell death when added to the cultures during the first 4 h post-exposure. They offered partial protection when added between 6 and 12 h and were of no benefit when added after 12 h post-exposure.

The determination and prevention of cytotoxic effects induced in human lymphocytes by the alkylating agent 2,2'-dichlorodiethyl sulfide (sulfur mustard, HD).

2,2'-Dichlorodiethyl sulfide (sulfur mustard, HD, 1,1'-thio-bis[2-chloroethane]) is a potent vesicant which can cause severe lesions to skin, lung, and eyes. There is no convenient in vitro or in vivo method(s) to objectively measure the damage induced by HD; therefore, a simple in vitro method was developed using human peripheral lymphocytes to study HD-induced cytotoxicity. The cytotoxicity of HD was measured using dye exclusion as an indicator of human lymphocyte viability. Exposure to HD resulted in both a time- and a concentration-dependent cytotoxic effect on human lymphocytes. Using this in vitro assay, the effectiveness of various therapeutics (niacin, niacinamide, and 3-aminobenzamide) in preventing HD-induced cytotoxicity was studied. Niacinamide and 3-aminobenzamide prevented the cytotoxic effects of HD for up to 2 days.

Comparison of cell size in sulfur mustard-induced death of keratinocytes and lymphocytes.

Sulfur mustard (HD) is a vesicant chemical warfare agent that directly alkylates cellular DNA and produces DNA strand breaks. To identify cellular models for in vitro screening of antivesicant compounds in DNA repair assays, we compared the mechanism of HD-induced cell death in cultured adult normal human epidermal keratinocytes (NHEK) and peripheral blood lymphocytes (PBL). One parameter that we used to distinguish apoptotic from necrotic cell death was the change in cell size due to HD. In the presence or absence of a poly(ADP-ribose) polymerase inhibitor (PARPI), cell preparations were exposed to various concentrations of HD (0.01-1.0 mM) and harvested at selected times after exposure (up to 24 h). Results from these experiments suggest that, with increasing HD concentration and time, NHEK will fragment irrespective of the presence or absence of PARPI, with cell fragmentation presumably preceded by necrosis. In the absence of PARPI, PBL size initially decreases and then remains constant over time. Previous DNA fragmentation studies indicate that both apoptosis and necrosis occur in HD-exposed PBL in a time-dependent manner. In the presence of PARPI, there is a HD concentration- and time-dependent decrease in PBL size that is characteristic of apoptosis. The shift in the mechanism of HD-induced PBL death from apoptosis followed by necrosis to exclusively apoptosis in the absence and presence of PARPI, respectively, is in agreement with previous findings on HD-induced changes in membrane integrity, energy levels and DNA fragmentation. Considering that NHEK fragment early after exposure to HD concentrations that produce vesication in human skin, PBL may be a more appropriate model for use in DNA repair assays.

Ultrastructural correlates of the protection afforded by niacinamide against sulfur mustard-induced cytotoxicity of human lymphocytes in vitro.

Sulfur mustard (HD) has been shown to cause a concentration-dependent decrease in viability of human lymphocytes in vitro as measured by dye exclusion; this decrease is preventable by inhibitors of poly(adenosine diphosphatase ribose) polymerase such as niacinamide. The present study investigates the morphologic correlates of the protection afforded by niacinamide through scanning and transmission electron microscopic analysis of human lymphocytes incubated in the presence or absence of 10(3) M niacinamide for 24 h at 37 degrees C and exposed in vitro to 10(-3) M HD. Lymphocytes exposed to HD alone demonstrated 30% to 40% viability and loss of microvilli, large cytoplasmic vacuoles, extensive blebbing of the perinuclear envelope, loss of cytoplasmic organelles, condensation of nuclear chromatin, and multiple perforations of the plasmalemma. In the presence of niacinamide HD-treated lymphocytes had a viability of 87% and, except for blunting of the microvilli, essentially normal ultrastructure. Although the sequence of observed ultrastructural changes was not established, results of this morphologic study suggest that, in addition to the prevention of plasmalemmal defects and dye infusion, the mechanism of niacinamide protection appears to include preservation of the morphologic and functional integrity of cellular organelles.

Effects of specific inhibitors of cellular functions on sulfur mustard-induced cell death.

This study was conducted to determine whether inhibitors of normal cellular functions can reduce cytotoxicity induced by sulfur mustard (HD). The compounds examined include inhibitors of poly(ADP-ribose) polymerase (PADPRP), inhibitors of mono(ADP-ribose) transferase (MADPRT), inhibitors of lipid peroxidation, and an inhibitor of protein synthesis. To determine the effects of these compounds on HD-induced cell death, human lymphocyte preparations were treated with known concentrations (0.1 microM to 1000 microM) of an inhibitor and exposed to an estimated 87% effect concentration (EC87) of HD (170 microM) for loss in cell viability. Cell viability was determined at 24-26 hr post-exposure to HD using a dye (propidium iodide) exclusion assay and a flow cytometer. All of the selected PADPRP inhibitors were found to be effective at reducing the cytotoxic effects of HD. These inhibitors were rank-ordered based on the concentration that gives 50% (EC50) reduction of HD-induced cell death. A significant correlation (r = 0.94) was observed between the compounds' ability to inhibit PADPRP and the compounds' ability to reduce HD- induced cell death, suggesting that PADPRP plays a role in HD-induced cell death. Inhibitors of MADPRT, lipid peroxidation, and protein synthesis were not effective at reducing HD-induced cell death.

Poly(ADP-ribose) polymerase inhibitors regulate the mechanism of sulfur mustard-initiated cell death in human lymphocytes.

Sulfur mustard (HD) produces slow-healing skin lesions that contain large, tight fluid-filled blisters. These lesions are the result of severe damage to areas of the body exposed to HD and require extensive medical care before complete recovery is achieved. Converting the mechanism of HD-initiated cell death from an inflammatory oncosis (homicide) to benign apoptosis (assisted suicide) may reduce the extent of cellular damage and the time required for healing. HD-exposed human lymphocytes lose cellular function, membrane integrity and viability, and suffer degradation of their nuclear components. The treatment of HD-exposed cells with poly(ADP-ribose) polymerase inhibitors prevents or alters the HD-initiated loss of cell viability, membrane integrity, cellular metabolic constituent (NAD) and cellular energy (ATP), while initiating alterations in nuclear constituents. It is hoped that by preventing or altering these HD-initiated changes we can limit the extent of the injury, decrease the time required for repair and reduce the loss of performance suffered by exposed individuals. The use of poly(ADP-ribose) polymerase inhibitors to assist in initiating apoptosis in affected cells should help to achieve these objectives while preventing the chance of further disease development later in the exposed individuals.

Simultaneous initiation of degranulation and inhibition of leukotriene release by soman in human basophils.

Previous studies noted that the serine esterase inhibitor, soman, could induce histamine release from human basophils. To investigate the mechanisms by which soman causes histamine release (a preformed mediator), we also examined its ability to induce leukotriene release (a newly synthesized mediator) from basophils. We found that no leukotriene release followed activation with soman, while histamine release was usually greater than 70%. In addition, soman and diisopropyl-fluorophosphate were found actively to suppress low level spontaneous leukotriene release as well as ongoing leukotriene release induced by anti-IgE antibody. Soman (0.3 mM) was able to stop leukotriene release as rapidly as the calcium chelator, EDTA. In a series of control experiments, it was noted that soman did not influence the metabolism of LTC4 to LTD4 or LTE4 (for which little metabolism occurred), eliminating the possibility that reduced LTC4 release could have resulted from its enhanced metabolism. Therefore, using one compound (soman), basophils could be simultaneously activated to degranulate while having the pathway leading to leukotriene release actively suppressed. These results provide further evidence that histamine and leukotriene release are independent pathways resulting from the activation of basophils.

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity.

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1-1000 microM HD, reduced NAD + levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 microM HD, NAD + levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD + depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a useful in vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.

Purified human mast cells and basophils release human elastase and cathepsin G by an IgE-mediated mechanism.

Human lung mast cells and human peripheral basophils were purified and examined for their content of proteolytic enzymes similar to lung Hageman factor activator (LHFA) previously found to be released from chopped human lung by an IgE-mediated mechanism. It was found that both cell types contained elastase and cathepsin G-like enzymes, and that elastase appeared to be responsible for LHFA activity. The enzymes, as well as histamine, were released in a dose-dependent manner from these cells by anti-IgE antibody and by ionophore A 23187. It appears that stimulation by either anti-IgE or ionophore increases the amount of both enzymes extractable from these cells. The release of these proteases may explain some of the inflammation and observed defects in coagulation which occur as a result of anaphylaxis.

Sulfur mustard lowers nicotinamide adenine dinucleotide concentrations in human skin grafted to athymic nude mice.

Human skin grafted to athymic nude mice shows a decrease in nicotinamide adenine dinucleotide (NAD+) concentrations when exposed to sulfur mustard (HD). The lowering of NAD+ is dependent on both dose of HD and time after exposure. When HD is applied to grafted skin at 127 micrograms/cm2, the decrease in NAD+ begins immediately after exposure, approaches minimal values by 4 hr. NAD+ returned to normal within 18 hr. With higher concentrations of HD, NAD+ concentrations fall precipitously within 4 hr, reach a minimum value at 18 hr, and seem to remain at this depressed value for at least 72 hr. NAD+ loss appears to precede and be proportional to tissue injury. Pretreatment of mice with 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, seems to lessen the effect of HD. At higher concentrations of HD, human grafts from mice pretreated with 3-aminobenzamide show significant protection from loss of NAD+ levels after 4 hr.