Anaphylatoxin-induced histamine release with human leukocytes: studies of C3a leukocyte binding and histamine release.

Purified human C3a and synthetic COOH-terminal peptides of C3a, i.e., a pentapeptide, Leu-Gly-Leu-Ala-Arg (5R), and an octapeptide, Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg (8R) induced histamine release from human basophil granulocytes. On a molar basis, 5R was one-tenth and 8R was one-fifth as active as C3a in causing histamine release. It was found that 125I-C3a binds to whole leukocytes, interacting with both mononuclear cells and neutrophils and the binding was inhibited by preincubation of cells with unlabeled C3a, but not by C5a. 5R and 8R also inhibited the binding of 125I-C3a to the cells. However, on a molar basis, 2,000 times more 8R or 6,000 times more 5R is required for 50% inhibition of 125I-C3a binding as compared with native C3a. Autoradiography of cells using 125I-C3a and 125I-C5a showed preferential binding of 125I-C3a to eosinophils and basophils, whereas 125I-C5a binds primarily to neutrophils and eosinophils and to a lesser extent to basophils. The preferential binding of C3a and C5a to different cell types may herald significance related to their physiological functions.

C5a-induced expression of P-selectin in endothelial cells.

Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.

Regulation of human neutrophil guanylate cyclase by metal ions, free radicals and the muscarinic cholinergic receptor.

We have examined the properties of soluble guanylate cyclase activity in the human neutrophil. The enzyme showed complex regulation by metal ions. A 10-fold higher activity was observed in the presence of Mn2+ than Mg2+, while Ca2+ caused an increase in activity only in the presence of Mg2+ ion. Sodium nitroprusside (SNP), azide and hydrogen peroxide were activators of the enzyme. Dithiothreitol blocked the activation by SNP, suggesting the involvement of thiol groups in the activation process. Carbachol acting through the muscarinic cholinergic receptor caused a dose-dependent activation, which was blocked by atropine. Higher concns of carbachol were required to activate guanylate cyclase than were required for the modulation of enzyme release elicited by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Nordihydroguaracetic acid inhibited carbachol stimulation of guanylate cyclase. By contrast, trifluoperazine (TFP), a calmodulin antagonist, caused a biphasic modulation of basal activity in the presence or absence of carbachol. Our results indicate that: allosteric interactions of metal ions are important to the regulation of the enzyme, the free radical nitroxide as well as hydrogen peroxide enhances enzyme activity, agonist occupancy of the muscarinic cholinergic receptor activates neutrophil guanylate cyclase probably through a mechanism involving calcium influx and the activation of the lipoxygenase pathway, and a TFP-sensitive site (possibly calmodulin) is involved in the selective regulation of basal enzyme activity.

Identification of receptor regulatory proteins, membrane glycoproteins, and functional characteristics of adenylate cyclase in vesicles derived from the human neutrophil.

Human neutrophils were disrupted by brief sonication under conditions which preserve the hormone sensitivity of adenylate cyclase and yield minimal granule lysis. Fractions enriched in adenylate cyclase were analysed for hormonal and guanine nucleotide regulation of the enzyme as well as structural proteins. Adenylate cyclase was activated by PGE1 and isoproterenol in a GTP-dependent fashion, while f-met-leu-phe and C5a gave no stimulation. Cholera toxin treatment, which specifically modifies cyclase-related GTP-binding proteins, caused a dose-dependent enhancement of GTP activation, in which GTP alone activated maximally and PGE1 was without further effect. The following proteins were detected in the cyclase-containing vesicles: a 42 K mol. wt protein labeled selectively by cholera toxin; protein subunits observed in SDS gels at 214, 165, 105 and 47 K, of which the 47 K band was the most prominent and comigrated with actin; prominent lectin-binding activities at 165 K (concanavalin A and wheat germ agglutinin) as well as at 100 K (wheat germ agglutinin); and a set of proteins and lectin-binding activities in fractions containing beta-glucuronidase activity distinct from adenylate cyclase containing vesicles. The identification of receptor-controlled cyclase, GTP-binding regulatory proteins, cytoskeletal elements and unique lectin-binding activities in a single vesicle preparation should contribute to an understanding of receptor-mediated control of neutrophil function.

Autoreactivity between lymphocytes and thymus cells in myasthenia gravis.

Thymus cell preparations from four of five myasthenia gravis patients with thymic hyperplasia and from one additional patient with thymic involution stimulated autologous peripheral blood lymphocytes. No autostimulation was observed in two patients with thymoma. Autostimulation as associated with an increase in the fraction of B lymphocytes in the thymus.

Activated complement in inflamed aqueous humor.

Activated complement is an important mediator of inflammation. Radioimmunoassay was used to measure levels of C3a, an activated fragment of C3, in aqueous humor. Additionally, immunoelectrophoresis was performed on aqueous humor to detect Factor B and its conversion product, Bb, as well as C3c, a breakdown product of C3. All six samples of normal aqueous humor had no detectable C3a, C3c, or Factor B. All eight samples of aqueous humor from patients with anterior uveitis had measurable levels of C3a. Factor B and C3c were detected in 3/7 samples of inflamed aqueous humor. Factor B was converted fully to Bb in two of these three samples, suggesting alternative pathway activation of complement. Activated complement fragments are present in the aqueous humor of eyes with anterior uveitis and may help mediate the inflammatory process.

Activation of the alternative complement pathway by intraocular lenses.

To determine if posterior chamber polymethylmethacrylate lenses with polypropylene loops activate complement, the authors measured levels of C3a, C4a and C5a by radioimmunoassay in human sera incubated with and without these lenses. Human sera incubated with intraocular lenses showed elevated levels of C3a and C5a but no change in C4a. There were no statistically significant differences in the generation of activated complement by polypropylene loops vs polymethylmethacrylate optics. The authors also compared the ability of intraocular lenses to activate complement with that of zymosan and endotoxin, known activators of the alternative pathway. Our results suggest that polymethylmethacrylate lenses with polypropylene loops generate C3a and C5a by activation of the alternative complement pathway.

Latex allergens in tire dust and airborne particles.

The prevalence and severity of latex allergy has increased dramatically in the last 15 years due to exposure to natural rubber products. Although historically this health risk has been elevated in hospital personnel and patients, a recent survey has indicated a significant potential risk for the general population. To obtain a wide-spread source for latex exposure, we have considered tire debris. We have searched for the presence of latex allergens in passenger car and truck tire tread, in debris deposited from the atmosphere near a freeway, and in airborne particulate matter samples representative of the entire year 1993 at two sites in the Los Angeles basin (California). After extraction of the samples with phosphate buffered saline, a modified-ELISA inhibition assay was used to measure relative allergen potency and Western blot analyses were used to identify latex allergens. The inhibition studies with the human IgE latex assay revealed inhibition by the tire tread source samples and ambient freeway dust, as well as by control latex sap and latex glove extracts. Levels of extractable latex allergen per unit of protein extracted were about two orders of magnitude lower for tire tread as compared to latex gloves. Western blot analyses using binding of human IgE from latex-sensitive patients showed a band at 34-36 kDa in all tire and ambient samples. Long Beach and Los Angeles, California, air samples showed four additional bands between 50 and 135 kDa. Alternative Western blot analyses using rabbit IgG raised against latex proteins showed a broad band at 30-50 kDa in all samples, with additional bands in the urban air samples similar to the IgE results. A latex cross-reactive material was identified in mountain cedar. In conclusion, the latex allergens or latex cross-reactive material present in sedimented and airborne particulate material, derived from tire debris, and generated by heavy urban vehicle traffic could be important factors in producing latex allergy and asthma symptoms associated with air pollution particles.

Hypoxic and Complement-Induced Lung Injury in the Isolated Perfused Rat Lung as Determined by Phosphorous-31 Nuclear Magnetic Resonance.

Magnetic resonance spectroscopy (MRS) is a non-invasive method of obtaining biochemical information. Patients or experimental animals are placed within a magnetic imaging device and specific patterns of chemical spectra, such as P31 patterns of adenosine triphosphate (ATP) can be recorded. We have injured the isolated rat lung by complement activation and hypoxia. Nuclear magnetic resonance patterns of phosphate metabolites revealed markedly diminished lung concentrations of ATP and phosphocreatine. In vivo measurement of lung metabolism through MRS may be a useful tool to measure metabolism of severe lung disease in man.

Comparative effect of C3a and C5a on adhesion molecule expression on neutrophils and endothelial cells.

Complement activation is known to enhance neutrophil binding to human umbilical vein endothelial cells (HUVECs). Recently, we have shown that recombinant human C5a upregulates P-selectin in HUVECs. Unstimulated human neutrophil binding is also increased on C5a stimulated HUVECs. We demonstrate in this report that C5a upregulates CD11b/CD18 in human neutrophils. Also shown is that synthetic C3a57-77 and an analog 15 amino acid C3a peptide (C3a15) neither upregulate CD11b/CD18 nor do the C3a peptides increase P-selectin, ICAM-1 or E-selectin in HUVECs. Thus C5a and not C3a is responsible for early (approximately 30 minutes) neutrophil adhesion to endothelial cells after complement activation.

Upper airways involvement in bronchial asthma.

The upper airways--the nose, pharynx, and mouth--lead through the larynx and into the tracheobronchial tree of the lung (the lower airways). This cavernous void in the upper airways transports external air to the alveolar sacs, in the distal segments of the tracheobronchial tree. Oxygen is absorbed from the alveolar sacs and carbon dioxide is released. Yet, under adverse physiologic conditions such as allergic or nonallergic rhinitis, sinusitis, and bronchitis, obstruction of the upper and lower airways occurs and leads to sneezing, rhinitis, and bronchospasm. The simultaneous occurrence of upper airways disease and asthma is addressed in this review.

Activation of human serum complement with allergens. I. Generation of C3a, C4a, and C5a and induction of human neutrophil aggregation.

To understand the relevance of allergens in generation of C3a, C4a, and C5a in normal human serum, we studied extracts of several allergens (house dust, house dust mite, Aspergillus fumigatus, and perennial ryegrass). Known complement activators zymosan and endotoxin were used as controls. Generation of C3a, C4a, and C5a (determined by radioimmunoassays) occurred with extracts of house dust and Aspergillus more than with house dust mite and ryegrass. Anaphylatoxins were produced both in dose and time-dependent fashions. Serum activated with house dust and Aspergillus extracts induced neutrophil aggregation when this serum was added to neutrophil suspensions. Less aggregation occurred when house dust mite and perennial ryegrass extract-activated sera were added to human neutrophils. We propose that some allergens may induce biologic responses by activation of sera and generation of anaphylatoxins as well as by IgE-mediated responses.

Complement activation requirements for histamine release from human leukocytes: influence of purified C3ahu and C5ahu on histamine release.

The activation of human sera for histamine release by zymosan and anti-BSA-BSA complexes at equivalence was dose dependent and associated with a marked fall in CH50, C3 and C5 hemolytic activities. Heat-aggregated IgG, though able to fix greater than 90% of human CH50 activity and produce a lesser fall in C3 and C5 activities than zymosan and BSA-anti-BSA complexes, was unable to induce basophil histamine release. Chemically purified human C3a and C5a each caused histamine release from human basophils; C5a was more potent than C3a. In addition, neither zymosan nor BSA-anti-BSA complexes released histamine when incubated with homozygous C3-deficient serum. The addition of C3a and C5a at suboptimal concentrations produced an additive effect of histamine release.

Chronic chyluria: a clinical study of 3 patients.

Studies of patients with chyluria or chylothorax have demonstrated significant disruptions of protein, blood and fat metabolism that may result in iron deficiency anemia, hypoproteinemia, hypolipidemia and malnutrition. To document the sequential development of these complications we performed serial clinical and biochemical studies for 2 to 12 years in 3 patients with presumed filarial chyluria whose sole treatment had been diethylcarbamazine. Despite the chronic loss of chyle in the urine these 3 patients did not have significant complications during the period of observation. The weight and blood pressure remained stable. No persistent anemia, hypoproteinemia or hypolipidemia was noted. Except for 1 patient in whom a transient decrease of the creatinine clearance developed during pregnancy, no permanent renal function impairment occurred. These observations suggest that chronic chyluria may not always result in serious alterations of the physical status or body functions of these patients requiring surgical repair, and supports the hypothesis that untreated chyluria could be a relatively benign process in our milieu.

HLA antigen studies in a family with C2 deficiency.

Two children in a family were found to be homozygous for C2 deficiency; both parents and a third child were heterozygous. C2 deficiency was associated with the HLA haplotypes carrying the antigens B18 and DW2. Antigen A10 was absent in this family. Mixed lymphocyte culture studies among the family members confirmed the association of C2 deficiency with the HLA-D locus.

Anaphylatoxin-induced neutrophil chemotaxis and aggregation. Limited aggregation and specific desensitization induced by human C3a and synthetic C3a octapeptides.

Human neutrophil aggregation was induced by highly purified human C3a and chemically synthetic COOH-terminal peptides of C3a (C3a-8R; Ala-Ala-Ala-Leu-Gly-Leu-Ala-Arg) in a dose-dependent manner and was 40% of human C5a-induced aggregation at each optimal concentration. In contrast to C5a and formyl-Met-Leu-Phe (f-MLP), C3a and C3a-8R showed little chemotactic activity. Specific desensitization of neutrophil aggregation was observed with C3a, C3a-8R, C5a and f-MLP, but not with C3a-des-Arg-7R, indicating that the human neutrophil has C3a-specific binding sites which are different from C5a and f-MLP receptors. An additive effect on aggregation was observed at suboptimal concentrations of C5a (1 X 10(-8) M) and C3a (1 X 10(-6) M) or C3a-8R (1 X 10(-5) M). These studies suggest that a subpopulation of human neutrophils have specific binding sites for C3a and C3a may work cooperatively with C5a during the process of neutrophil activation by increasing aggregation and lysosomal enzyme release.

Particulate air pollution: possible relevance in asthma.

The relative importance of air pollution in the pathogenesis of bronchial asthma has been of interest for several decades. Numerous studies on the role of gaseous air pollution containing ozone, nitrogen dioxide, sulfur dioxide, and carbon monoxide have been published. Very little attention has been focused on the role of respirable particles in the causation of asthma. In this article we summarize some of our ongoing investigations into the sources and composition of airborne particles in the Los Angeles and Pasadena atmosphere, including the search for biologically active particles that may induce asthma attacks. If is found that the urban atmosphere contains not only combustion-derived particles from diesel engine exhaust and gasoline-powered motor vehicle exhaust, but also particles formed from biological starting materials including plant debris, cigarette smoke, wood smoke, and meat smoke as well as tire debris containing some natural rubber and paved road dust. Paved road dust is a very complex mixture of particles including garden soil, tire dust, plant fragments, redeposited atmospheric particles of all types, and pollen fragments presumably ground up by passing traffic. We have shown previously that latex allergen can be extracted from tire dust, from roadside dust, and from respirable air samples taken at Los Angeles and Long Beach. At present, work is underway to identify the larger range of allergens that may be contributed by the entrainment of paved road dust into the atmosphere. The possible importance of pollen fragments present in paved road dust in very small particle sizes is discussed as well as their potential relevance in asthma.

Quantitative requirements for C3 to induce Forssman systemic shock and cutaneous hemorrhagic vasculitis in guinea pigs.

The requirements of complement (C) to induce systemic and cutaneous Forssman reactions were studied in inbred DHC-BA and Hartley strain guinea pigs. After intravenous injection of Forssman antibody, fatal systemic shock was associated with a marked drop in CH50, C4, and C3 and a lesser decrease in C5 hemolytic activity. Platelet counts and leukocyte counts dropped as well. With the use of the purified low molecular weight factor from cobra venom (CVF) to deplete C3, guinea pigs with less than 1% intravascular C3 were protected from lethal shock. Approximately 1% to 3% C3 activity is required for Forssman cutaneous vasculitis. These results confirm earlier studies that classical complement pathway activation occurs in Forssman shock and demonstrate the exquisite biologic efficiency of C3 in provoking the shock syndrome.

Effect of maternal immunotherapy on immediate skin test reactivity, specific rye I IgG and IgE antibody, and total IgE of the children.

The effect of specific immunotherapy during pregnancy was studied in 14 children, 3 to 12 years after delivery. Fourteen additional children from the same allergic mothers, in whom immunotherapy was not given during the pregnancy, served as controls. The immediate skin test response to grass allergens of the children of mothers given immunotherapy. Levels of rye I IgG and total IgE were lower in the sera of children born to mothers who received immunotherapy (not statistically significant) than their control cohorts. Paired cord blood and maternal blood samples drawn at delivery showed similar levels of rye I IgG, indicating that blocking antibody freely crosses the placenta. This evidence indicates that immunotherapy during pregnancy may have an inhibitory effect on immediate skin reactivity to grass allergens in some of the offspring. Whether tolerance to other allergens can be induced in children by maternal immunotherapy remains to be determined.