Methods to detect and quantitate complement activation.
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Antigen-Antibody Complex↗
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[Immunohistochemical C5b-9-complement complex demonstration in early stages of myocardial necroses using paraffin sections].
With conventional staining techniques it was nearly impossible to differentiate with certainty between early ischemic myocardial lesions, autolytic changes and intact myocardium without a nuclear section. In 1986 it was introduced an immunohistochemical method, which presented C5b-9 Complement Complex as a necrosis marker in cryostat sections. Our further development of this process has resulted in a technique for application on routine paraffin sections: C5b-9 Complement Complex is particularly well-suited for the exact and autolytically insensitive presentation of disseminated necroses of fiber bundels and single fibers of myocardium. The application of the technique on paraffin sections will lead to the use of the same in forensic medicine routine diagnostics of early ischemic myocardial lesions, even in cases of "sudden" cardiac death.
Complement C5↗
Isolation of three separate anaphylatoxins from complement-activated human serum.
Recent methodologies used in preparing anaphylatoxins from complement-activated serum are described. Activation of the alternative pathway generates C3a and C5a; however, activation of the classical pathway is required to generate the anaphylatoxin from C4. This article describes an activation scheme that simultaneously generates all three of the anaphylatoxins (e.g., C3a, C4a and C5a) in human serum and outlines a procedure for isolating each as homogeneous products. Purification of intact anaphylatoxins directly from complement-activated serum takes place only if an exopeptidase in serum, known as carboxypeptidase N (SCPN), is properly inhibited. A new series of mercapto derivatives of arginine analogs are introduced as potent and effective inhibitors of SCPN. These inhibitors permit normal complement activation but prevent degradation of the released activation fragments C3a, C4a or C5a. The SCPN inhibitor previously used was 6-aminohexanoic acid (EACA), but it required a 1 M concentration for effective inhibition, the substituted mercapto-guanido compounds prove to be effective in the mM range.
Aminocaproic Acid↗
Detection of complement activation in human serum using C5a-induced chemiluminescence of human granulocytes.
The complement system can be activated by many factors, including immune complexes, leading to the generation of biologically active split products like C5a anaphylatoxin. This study presents a technique which may be used for measuring C5a activity in human serum. The tetanus-anti-tetanus immune complex and aggregated human IgG were used as model activators of the complement cascade. The C5a activity was measured by C5a-induced chemiluminescence of granulocytes; furthermore, a radioimmunoassay was used to detect the C5a peptide. There was a strongly positive correlation between the two assay systems. The described method should be useful as an alternative means of detecting complement activation in the serum of patients with inflammatory diseases.
Antigen-Antibody Complex↗
Chemotaxis: basic aspects of methodology, mechanisms and pathology.
This review is an attempt to discuss the basic conceptual tools that are a prerequisite for any clinical study of chemotactic defects. They include familiarity with definitions of common terms and with the merits and drawbacks of the several possible in vivo and in vitro assay methods. Cellular mechanisms involved in locomotion are complex. They include basic requirements for cell metabolism as well as receptor recognition, attachment to surfaces and contraction of the cytoskeleton of the cell. Of the many chemotactic factors reported, few are well characterized and universally agreed upon. Similarly, with the use of more stringent criteria, a number of clinical defects of chemotaxis have proven transitory or even artifactual.
Adult↗
Analysis of the proinflammatory property of epidermal cyst contents: chemotactic C5a anaphylatoxin generation.
We investigated in vitro the contents of epidermal cysts for complement activation and found that they activated complement mainly through the alternative pathway. Chemotactic C5a anaphylatoxin produced by the cyst contents after contact with serum most likely plays a significant role in the initiation and aggravation of inflammation in ruptured epidermal cysts. Our additional study disclosed that components of three representative follicular resident microorganisms (Pityrosporum ovale, Propionibacterium acnes, and Staphylococcus epidermidis) also produced C5a anaphylatoxin mainly through the alternative pathway; the C5a production was more vigorous than that by a virulent pathogen, Staphylococcus aureus. These results suggest that accidental colonization of the cyst contents by these follicular microbial flora further augments the inflammatory changes in ruptured epidermal cysts.
Adult↗
Epidermal lymphocyte chemotactic factor specifically attracts OKT4-positive lymphocytes.
Epidermal lymphocyte chemotactic factor (ELCF) from skin overlying a positive tuberculin reaction was compared with the chemoattractants leukotriene B4 (LTB4), N-formyl-methionyl-leukyl-phenylalanine (FMLP), and complement split product C5a (C5a). The chemotactic assay used is a modified Boyden chamber technique. The lymphocytes were subsets of T lymphocytes from healthy young individuals first separated by flotation of E rosettes on Isopaque Ficoll followed by incubation of T cells with anti-CD4 and anti-CD8 monoclonal antibodies and further separation using fluorescence-activated cell sorting. ELCF specifically attracted OKT4+ lymphocytes, while LTB4, FMLP, and C5a induced significant migration in both OKT4+ and OKT8+ lymphocytes without any clear difference between the various chemoattractants or cell populations. We found no blocking of the chemotactic capacity of ELCF when we added antibodies towards IL-1 alpha and IL-1 beta to the chemotactic assay. Further recombinant IL-1 alpha and Il-1 beta did not induce any chemotactic response. Our observation may be of significance in explaining the predominance of OKT4+ cells in allergic contact dermatitis and certain other skin diseases.
CD4-Positive T-Lymphocytes↗
Involvement of complement in psoriasis and atopic dermatitis--measurement of C3a and C5a, C3, C4 and C1 inactivator.
Normal complement components and activation products were determined in the peripheral blood of 35 patients with atopic dermatitis (AD) and 24 patients with psoriasis at a mild to intermediate stage. None of the patients had received systemic or local steroid therapy 6 weeks prior to blood collection. Levels of C3, C4 and C1 inactivator (C1 INA) were determined in serum by radial immunodiffusion, whereas C3a and C5a levels were measured by radioimmunoassay. In comparison to healthy non-atopic controls, the levels of C3, C4 and C1 INA were found to be significantly increased in both diseases. No substantial differences were detected between patients with psoriasis vulgaris and psoriasis guttata, which suggests that the dissimilarities found were not due to preceding or concomitant infections. In AD, there was a tendency towards increased C3a levels, whereas in psoriasis, C3a levels were significantly increased. In both diseases, no measurable amounts of C5a could be detected. The results indicate that, in both AD and psoriasis, the complement participates in the inflammatory process. Elevated levels of C3a suggest that there is a continuous activation of the complement system leading to the generation of inflammatory mediators.
Adolescent↗
Induction of platelet aggregation by the complement-derived peptides C3a and C5a.
The effect of two complement-derived peptides, hog serum C3a and C5a, on platelet aggregation in platelet-rich plasma and suspensions in Tyrode solution was investigated. 1. Guinea-pig platelets were aggregated by both C3a and C5a; the spasmogenically inactive product of C3a, C3ai, also induced aggregation. Threshold concentrations were in the range of 10(-6)--10(-9) M depending on the peptide and platelet preparation. 2. Cat platelets were aggregated by C5a (threshold concentrations 10(-7)--10(-8) M) but not by C3a. 3. Platelets from pig, rabbit and man were not aggregated by either of the two peptides in concentrations of up to 5 X 10(-6) M. 4. When C5a was administered repeatedly in subthreshold doses guinea-pig platelets became tachyphylactic to C5a but were still aggregable by C3a or ADP. Conversely, platelets desensitized to C3a still reacted to C5a or ADP. Tachyphylaxis towards C5a developed also when platelets were incubated with C5a in the absence of free Ca2+ under which condition they do not react. The tachyphylaxis in this case became evident after recalcification of the medium. The lack of cross-desensitization indicates that C3a and C5a react via different receptors. 5. C3a and C5a were injected i.v. into guinea pigs. Histological examination of the lungs revealed that some of the smaller vessels (20-30 mu in diameter) were occluded by platelet aggregates. In addition signs of severe acute emphysema were seen in animals treated with C5a, but only slight emphysema in C3a-treated animals. Intravenous injections of C3a into guinea pigs caused but weak respiratory distress and drowsiness and never killed an animal (at doses of up to 20 mg per kg body weight), whereas C5a caused the well-known severe respiratory failure and death already at doses of 0.03 mg/kg body weight.
Anaphylatoxins↗
Comparative study on biological activities of various anaphylatoxins (C4a, C3a, C5a). Investigations on their ability to induce platelet secretion.
Several anaphylatoxic substances (human C3a, guinea pig C3a, human C4a, guinea pig C5a, and a synthetic C3a-related hexapeptide) were compared with regard to their ability to induce secretion of [3H] serotonin from guinea pig platelets. Functional identity of the C3a preparations, C4a, and the hexapeptide was demonstrated by the phenomenon of crossed desensitization. Whereas C3a of human and guinea pig origin proved to be qualitatively and quantitatively identical, C4a expressed only 3% of the activity of the C3 fragments on a molar basis. Investigations with goat anti-guinea pig C3a demonstrate that human and guinea pig C3a possess one antigenic determinant in common; however, this determinant is not the C-terminal amino acid sequence. Addition of the anaphylatoxins with low doses of thrombin led to a potentiation of [3H] serotonin release from the platelets. Under these conditions C3a concentrations of 1.5 X 10(-10) mumol/liter (65 pg of C3a) could be detected. Thus the platelet system represents the most sensitive in vitro assay known for evaluation of biological activity of the C3a anaphylatoxins.
Anaphylatoxins↗
Stimulus interactions in release of superoxide anion (O2-) from human neutrophils. Further evidence for multiple pathways of activation.
A wide variety of agents stimulate superoxide anion (O2-) release from human neutrophils. To determine whether the same or different cellular pathways are utilized, neutrophils were stimulated to release O2- with combinations of f-Met-Leu-Phe (FMLP) (10(-7)M), C5a (25 nM), Con A (100 micrograms/ml), arachidonic acid (100 microM), and PMA (1 microgram/ml). These concentrations produced maximal O2- production when used alone. A synergistic response was observed when Con A was used in combination with FMLP or C5a. This response was twice the expected release in cytochalasin B-treated cells and three to five times the expected release in untreated cells. Additional studies showed that synergism was dependent upon the simultaneous presence of both agents. Additive O2- responses were observed when either FMLP, Con A, or C5a was tested in combination with arachidonic acid and when FMLP and C5a were tested together. When PMA was tested with C5a, FMLP, or Con A, a nonadditive O2- response resulted, whereas mixtures of PMA and arachidonic acid resulted in a less than additive response. These contrasting results using different soluble stimuli in combination suggest that multiple pathways exist for the stimulation of neutrophil O2- release, with some stimuli being totally independent of each other (possibly activating separate pools of oxidase), while other stimuli show cooperative effects on oxidase activation.
Adult↗
Chemotaxis of polymorphonuclear leukocytes in response to surface-bound complement-derived chemoattractants generated in situ.
Polymorphonuclear leukocytes (PMNLs) were allowed to migrate on slides with fixed yeast particles dotted about on the surface. Locomotion was quantified by counting the number of yeast particles in association with a PMNL. Yeast particles that differed in their ability to consume hemolytic complement differed also in their ability to generate chemoattractants. Addition of a complement source to yeast particles able to activate the complement system resulted in a chemotactic response, as well as when fluid-phase attractants were removed prior to the measurement of PMNL chemotaxis, indicating that the chemoattractants generated were absorbed to the surface. Using an immunofluorescence technique, it was found that complement factor 5 coated a circular area around each yeast particle, provided that the particles were able to activate the complement system.
Chemotactic Factors↗
Endotoxin tolerance diminishes certain antiinflammatory effects of endotoxin.
Endotoxin (bacterial lipopolysaccharide, LPS) is paradoxically both inflammatory and antiinflammatory. A single intravenous injection of 100 micrograms Escherichia coli LPS markedly inhibits the inflammatory changes associated with cutaneous reversed passive Arthus (RPA) reactions in New Zealand white rabbits. Polymorphonuclear (PMN) leukocytes from LPS-treated rabbits exhibit diminished responsiveness in vitro to complement (C5) -derived peptides. Repeated injections of LPS render animals "tolerant", that is, refractory to the toxic and inflammatory effects of LPS. We examined whether tolerance would enhance the ability of LPS to inhibit inflammation not attributable to LPS. Surprisingly, as compared with rabbits receiving a single dose of LPS, tolerant rabbits demonstrated greater inflammatory changes (i.e., PMN exudation, vascular permeability) associated with RPA reactions. PMNs from LPS-tolerant rabbits responded in vitro to C5-derived peptides significantly more than PMNs from rabbits that received a single dose of LPS. We speculate that some antiinflammatory effects of LPS require the toxic or inflammatory effects of LPS itself. These observations might relate to the limited efficacy of fever therapy and the variable effects of gram-negative sepsis on functions of human PMNs.
Animals↗
Inhibition of human neutrophil secondary granule discharge by antiinflammatory agents.
Human neutrophil cobalamin binding protein (NCBP) is located exclusively in the neutrophil secondary granules. The soluble stimuli formlymethionyl-leucyl-phenylalanine and the low-molecular-weight complement fragment C5a both promote the dose-dependent release of NCBP from cytochalasin B-treated neutrophils in vitro. The extracellular discharge of NCBP induced by higher secretagogue is inhibited by prior exposure of neutrophils to the corticosteroids hydrocortisone and methylprednisolone and the nonsteroidal antiinflammatory agents indomethacin and ibuprofen. The four antiinflammatory agents function as competitive antagonists of neutrophil secondary granule discharge with a site of action at or near the cell surface. These findings support the hypothesis that antiinflammatory agents prevent neutrophil activation in vitro by inhibition of stimulus-receptor coupling. The significance of these observations with regard to the in vivo actions of these agents remains uncertain, however.
Anti-Inflammatory Agents↗
Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes.
The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.
Animals↗
Acquired c1-inhibitor deficiencies in lymphoproliferative diseases with serum immunoglobulin abnormalities. A study of three cases.
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Aged↗
Genetic factors in host resistance to urinary tract infection.
In patients with recurrent pyelonephritis, the pathogenetic events proceed through intestinal colonization, spread to the urinary tract and persistence, seemingly uninterrupted by host defense mechanisms. The factors responsible for the deficient bacterial clearence from the kidneys of these patients, and the genetic control, have not been identified. The susceptibility to colonization has been linked to an increased receptivity for attaching bacteria of the uroepithelia, and to an overrepresentation of the P1 blood group phenotype. To evaluate the role of defects in host defense for the susceptibility to pyelonephritis, experimental UTI in mouse strains with known deficiencies was used. A highly significant increase in susceptibility was noted for C3H/HeJ compared to C3H/HeN mice. The bacterial recovery was inversely correlated to the mitogenic response to LPS. Back-cross analysis revealed a linkage of susceptibility to the Lpsd/Lpsd genotype. In contrast, T and B lymphocyte and complement (C5) defects had little effect on the clearance of Escherichia coli from the kidneys. It is concluded that the inflammatory mechanisms induced by LPS are essential for resistance to experimental pyelonephritis.
Animals↗