Differentiation driven by granulocyte-macrophage colony-stimulating factor endows microglia with interferon-γ-independent antigen presentation function

The antigen presentation function of microglial cells was analyzed after differentiation in neonatal mouse brain cell cultures supplemented either with macrophage (M) or granulocyte/macrophage (GM) colony-stimulating factor (CSF). The cells separated from concomitant astrocytes in both culture systems turned out to exhibit cytological characteristics of macrophages and bore MAC-1 and F4/80 markers in a similar way. When comparatively tested for accessory cell function, only microglia developed with GM-CSF were able to efficiently induce antigen-directed proliferation of a series of helper T cell lines representing both the TH1 and TH2 subtype. Antigenic T cell activation by this microglia p…

The Anaphylatoxic Peptide C3a of Guinea Pig Complement

Abstract Highly purified guinea pig C3a was obtained after specific cleavage of isolated C3 by the alternative pathway enzyme VF-B in a one step procedure. It turned out to be a low molecular weight peptide with basic character (M.W. 9500; isoelectric point above 9.4). C3a represents an antigenetic determinant of its own in the native C3 molecule, different from the B determinant. Guinea pig C3a is resistant to 100°C for 10 minutes. Its smooth muscle contracting activity can be destroyed by trypsin and carboxypeptidase B. These findings indicate that guinea pig C3a is quite similar to human C3a.

Stimulation of PGE and TXB2 release from macrophages by C3b

Morphological changes in adherent cells induced by Clostridium difficile toxins.

Activation of the alternative pathway of complement: efficient fluid-phase amplification by blockade of the regulatory complement protein β1H through sulfated polyanions

Current concepts of activation of the alternative pathway of complement (APC) focus on the central role of an amplification mechanism triggered by C3b which is covalently bound to the surfact of activating substances. Using sulfated polyanions as model substances, an efficient fluid-phase activation of complement is demonstrated in contrast to solid-phase activation. It is shown that particulate high-molecular weight sulfated polyanions are capable of reversible binding the guinea pig and human regulatory protein beta1H. This fixation leads to an extensive activation of C3 and factor B because the regulatory function of beta1H is blocked in the fluid-phase C3b-dependent amplification system…

An inherited deficiency of the third component of complement, C3, in guinea pigs

Hereditary deficiency of the third component of complement, C3, is found very seldom in the human. C3 deficiency is associated with severe bacterial infections revealing the central role of C3 in complement activation via the classical or alternative pathway. We describe a new hereditary C3 deficiency in strain 2 guinea pigs. Serum from these animals had a markedly reduced lytic activity in a standard assay for complement-dependent, antibody-mediated cytotoxicity. In functional assays of individual components, the hemolytic activity of the components C4, C2, C5 and of factors B, D and H was in the normal range. The functional C3 titer, and similarly C3 antigenic activity in the serum of the…

Ability of the T cell-replacing polyanion dextran sulfate to trigger the alternate pathway of complement activation.

Dextran sulfate (DS) consumed C3 in C4 deficient guinea pig serum. This temperature-dependent reaction required Mg++ ions and could therefore be blocked by EDTA. Isolated C3 was not influenced by DS, but serum factors were required for C3 consumption. The C3 proactivator as well as C3 were converted to their activated state by DS in guinea pig and human serum, as revealed by immunoelectrophoretical analysis. DS generated anaphylatoxin activity in serum. It is concluded that DS activates C3 via the alternate pathway of complement activation. This potency of the polyanion might serve as a tentative explanation for its T cell-replacing effect in an antibody-forming system, which was reported b…

Activation of macrophages by platelet activating factor (PAF)

Complement components in relation to macrophage function

Effect of in vivo stimulation of mice on the secretion of factor B of the alternative complement pathway by peritoneal macrophages

After in vivo treatment of mice with thioglycollate medium, the amount of native factor B which could be detected in vitro in culture supernatants of peritoneal macrophages was much lower than that found in supernatants of macrophages taken from untreated mice. However, when the macrophages from thioglycollate medium-treated mice were cultured on a plastic surface covered with glutardialdehyde-linked bovine serum albumin, the culture supernatants contained larger quantities of native factor B than culture supernatants of macrophages from untreated mice under the same conditions. Thus, the effect of in vivo thioglycollate medium treatment on the in vitro secretion of factor B by peritoneal m…

Anaphylatoxic peptide C3a of complement and synthetic hexapeptide C3a 72–77 evoke thromboxane generation by macrophages

Formation and function of a complement-activating enzyme generated from factors of guinea pig serum and cobra venom

An enzymatic complex can be formed by factors from guinea pig serum and cobra venom, which is able to activate C3 bypassing C1, C4 and C2. Formation and action of the enzyme are described. The action on C3 results in an activation of the terminal complement components and in membrane destruction provided suitable membrane receptors are available.

Suppressive effects of C3b on monocyte-dependent T cell proliferation.

The effect of C3b treatment of human monocytes on secondary antigen-dependent T cell response was studied. When antigen-specific T cell blasts were cultivated together with C3b-treated monocytes the proliferative response was inhibited in a dose-dependent fashion. This suppressive effect was specific for C3b because heat-inactivated C3b or buffer alone had no influence on T cell proliferation. In part, this suppressive effect is mediated through a C3b-induced decreased expression of class II antigens on the surface of treated monocytes, but another suppressive mechanism exists because the C3b pretreatment of monocytes also led to an inhibition of the proliferative response in a class II ant…

In vitro synthesis of factor B of the alternative pathway of complement activation by mouse peritoneal macrophages

Factor B of the alternative pathway of complement activation was shown to be synthesized and secreted by unstimulated mouse peritoneal macrophages. The activity of B in the culture supernatants from macrophage monolayers was detected by consumption of C3 in reaction mixtures containing supernatant and guinea pig factors C3, D and insoluble C3b. Using a monospecific antiserum, factor B in concentrated culture supernatants was shown by immunodiffusion and immunoelectrophoresis to be identical to factor B in mouse plasma and to form a characteristic complex with cobra venom factor in the presence of D. A steady rate of factor B secretion was observed for 4 days providing the medium was changed…

Linkage of C4 and C4 deficiency to BF and GPLA

The C4, Bf, and GPLA phenotypes of homo- and heterozygous C4-deficient guinea pigs were studied. The electrophoretic patterns suggest that the deficiency in circulating C4 results from an impaired structural gene, allelic to the C4F, C4S, and C4S1 alleles at the C4 locus. In family studies, support for linkage of C4 and Bf to theGPLA system was obtained. The defective gene appears to be the fourth allele, which is rare, in the polymorphism of the fourth component of guinea pig complement.

Comparative study on biological activities of various anaphylatoxins (C4a, C3a, C5a)

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 determi…

Involvement of phospholipid metabolism in the events of the oxidative burst of granulocytes

In summary we provide now evidence for specific interactions between the phospholipid metabolism and the oxidative burst. Lysophosphatidylcholine-like molecules showed specific enhancement of the oxidative burst and the capacity to replace phospholipase A2 function. Our findings might be useful in further studying the events occurring on the way from receptor stimulation to activation of the oxidative burst.

Physicochemical characterization of the fifth (C5), sixth (C6), seventh (C7), eighth (C8) and ninth (C9) component of guinea pig complement.

A physicochemical characterization of the purified guinea pig complement components C5 to C9 is given. For this purpose the sedimentation rate, the diffusion coefficient, the molecular weight and the isoelectric point were determined and compared with the values already known for the guinea pig and human complement system. For the determination of the physicochemical parameters gel filtration on Sephadex G-200, ultracentrifugation applying a sucrose density gradient and thin-layer isoelectric focusing were used. By comparing the values of the human and guinea pig complement a remarkable similarity is shown.

COMPLEMENT-DEPENDENT B-CELL ACTIVATION BY COBRA VENOM FACTOR AND OTHER MITOGENS?

It has been proposed that two distinct signals are required for the triggering of the precursors of antibody-forming bone marrow-derived cells (B cells): (a) the binding of antigen or of a mitogen to the corresponding receptor sites on B-cell membranes and (b) the interaction of activated C3 with the C3 receptor of B lymphocytes. There is growing evidence that B-cell mitogens and T (thymus-derived cell)-independent antigens are capable of activating the alternate pathway of the complement system (bypass). Therefore, the effect of another potent bypass inducer was investigated with regard to B-cell activation and the role of C3. Purified, pyrogen-free cobra venom factor was mitogenic for bot…

Demonstration of High-Affinity Binding Sites for C3a Anaphylatoxin on Guinea-Pig Platelets

3H-serotonin release from guinea-pig platelets was demonstrated to be the consequence of C3a binding to these cells. A Scatchard analysis of dose-response data of the 125I-C3a binding pattern to guinea-pig platelets pointed to the existence of binding sites with high and low affinity for the C3a molecule (HA and LA receptors). HA receptors are specific for C3a with intact C-terminal arginine. whereas C3adesarg only interacts with LA receptors. The release of serotonin may be induced by a combined reaction of C3a with HA receptors and LA receptors on the platelet membrane.

Comparative study on biological effects of the guinea pig complement-peptide C3a and C3a-related synthetic oligopeptides

Dose-response experiments with guinea pig C3a and a synthetic hexapeptide (amino acid residues 72–77), representing the COOH-terminal sequence of human C3a, were performed in two recently described bioassay systems for C3a, i.e. cytotoxicity against tumor cells measured as LDH and 51Cr-release and non cytolytic serotonin release from guinea pig platelets. Compared to the classical anaphylatoxic assay (guinea pig ileum contraction), nearly identical reactivities were observed in all three test systems with C3a and, although quantitatively different, with hexapeptide.

Platelet Activation: a New Biological Activity of Guinea-pig C3a Anaphylatoxin

3H-serotonin-release from labelled gp-platelets is established as a sensitive method for testing a new biological activity of gp-C3a anaphylatoxin in an autologous situation. Time-, dose- and temperature-dependent release reactions as well as specific inhibition by carboxypeptidase B and anti-C3a antibodies show that C3a is a potent and specific inducer of platelet activation. Inactive C3a does not induce 3H-serotonin-release but specifically inhibits the action of C3a on platelets.

Cloning and Characterization of Overlapping DNA Fragments of the Toxin A Gene of Clostridium difficile

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of vari…

Quantitative studies of the secretion of complement component C3 by resident, elicited and activated macrophages. Comparison with C2, C4 and lysosomal enzyme release.

To quantitate the secretion of complement component C3 by guinea pig peritoneal macrophages an enzyme-linked immunosorbent assay was developed. C3 secretion was studied in resident, elicited and activated macrophages and compared with release of hemolytically active C2 and C4, as well as the lysosomal enzyme β-D-2-acetamido-2-deoxyglucosidase. Resident macrophages secreted about 6 ng C3/106 cells/h into culture supernatants over a period of 12 h. Corynebacterium parvum-activated cells were found to secrete 3 times that amount at nearly constant rates. There was a stepwise increase in secretion of functional C2 and C4 when comparing resident, elicited and activated macrophages; secretion was…