Counterregulatory effects of interferon-gamma and endotoxin on expression of the human C4 genes.

Susceptibility to autoimmune disease is associated with null alleles at one of the two genetic loci encoding complement protein C4. These two genetic loci, C4A and C4B, are highly homologous in primary structure but encode proteins with different functional activities. Expression of C4A and C4B genes is regulated by IFN-gamma in human hepatoma cells and in murine fibroblasts transformed with the respective genes. In these cell lines, IFN-gamma has a significantly greater and longer-lasting effect on expression of C4A than that of C4B. In this study we examined synthesis and regulation of C4A and C4B in peripheral blood monocytes from normal, C4A-null, and C4B-null individuals. Synthesis of C4 in human peripheral blood monocytes decreases during time in culture. IFN-gamma mediates a concentration- and time-dependent increase in steady-state levels of C4 mRNA and a corresponding increase in synthesis of C4 in normal human monocytes. LPS decreases monocyte C4 expression and completely abrogates the effect of IFN-gamma on the expression of this gene. In contrast, LPS and IFN-gamma have a synergistic effect in upregulating expression of another class III MHC gene product, complement protein factor B. The effect of LPS on constitutive and IFN-gamma-regulated C4 synthesis is probably not mediated via release of endogenous monokines IL-1 beta, TNF-alpha, or IL-6. Synthesis of C4, and regulation of its synthesis by IFN-gamma and LPS, are similar in normal, C4A-, and C4B-null individuals. These results demonstrate the synthesis of C4 at extrahepatic sites and tissue-specific regulation of C4 gene expression.

Interaction of C3 and C3b with immunoglobulin G.

Human C3b as well as native C3 were found to bind to solid phase human and rabbit IgG. Haemolytically active C3 had significantly higher binding capacity to IgG than the C3b fragment. Inhibition experiments proved that C3 and C3b have common binding sites on the Fab and Fc part of the IgG molecule but the character of these binding sites was different. As a functional consequence of C3-IgG interaction, C3 binding was found to inhibit the specific precipitation of an IgG antibody preparation.

Complement-mediated fragmentation of soluble and insoluble immune complexes containing porcine anti-DNP antibodies.

Complement-mediated release of soluble immune complexes and immune precipitates containing DNP-PSA and precipitating or non-precipitating porcine anti-DNP antibodies was studied. A decrease in the average size of soluble immune complexes indicating their fragmentation was observed during incubation in excess human serum, the extent of the complex release was found to be in direct proportion to the time of incubation. The effect was complement-dependent. In the second part of the study, complement-dependent solubilization of the immune precipitates of the precipitating antibody preparation was compared to the solubilization of the precipitates of the non-precipitating antibody formed in the presence of PEG. Although, both types of precipitates activated complement in about the same extent, complexes of non-precipitating antibody were solubilized much slower than those of the precipitating one. As avidity of both antibody preparations to the antigen was high, the observed differences in the rate of the complex solubilization probably reflected differences in the structure of the two types of complexes.

The quaternary Gs3 and Gs7 allotypes of the rabbit: generation of the determinants by interspecies molecular hybridization.

A number of allotypic markers of the rabbit immunoglobulins are present only on intact immunoglobulin molecules and not on the isolated heavy or light chains. Some of these markers, although determined by the kappa light chain, are limited to a single heavy chain class of Ig. They can be generated by in vitro hybridization of the kappa light chain with the appropriate heavy chain. The quaternary IgG allotypic determinants Gs3 and Gs7 which are determined respectively by the b4.1 and b4.2 allelic variants of the kappa b4 light chain can be generated not only by in vitro hybridization of these light chains with a rabbit gamma chain, but also to a certain extent by their hybridization to gamma chains derived from other species.

The effect of lactoferrin on complement mediated modulation of immune complex size.

Earlier we showed that human lactoferrin is capable of inhibiting the complement-mediated lysis of antibody-coated erythrocytes due to an inhibition of the formation of the classical C3 convertase. In this report the effect of lactoferrin on complement mediated modulation of immune complex size was studied. Lactoferrin was shown to inhibit the complement-dependent solubilization of immune precipitates with kinetics resembling that seen by others using C2 deficient serum. Lactoferrin, however, did not affect the complement-dependent inhibition of immune precipitation. These findings confirm our earlier observations that lactoferrin has no effect on the activation of the very early complement components (C1 and C4), but inhibits C3 deposition on immune complexes which is a crucial mechanism in immune precipitate solubilization.

The immunomodulatory effect of human IgG Fc fragments on the oxazolone-specific immune response of high and low responder mice.

Intravenous injection of human IgG Fc fragments in mice resulted in the stimulation or inhibition of an oxazolone-specific antibody response depending on the schedule of Fc fragment injection. High and low responder mice for oxazolone were injected with Fc fragments according to two protocols: either on the day of oxazolone priming, or together with the oxazolone boost, and the isotype composition of oxazolone-specific antibodies was analysed by solid phase radioimmunoassay. We found the primary and secondary anti-oxazolone IgM levels increased in all instances, irrespective of the schedule of Fc fragment treatment. In contrast, the oxazolone-specific IgG production was increased only if Fc fragments were injected at the time of antigen priming. Injection of Fc fragments together with a secondary injection of oxazolone resulted in the inhibition of oxazolone-specific IgG production. Both stimulation and inhibition of oxazolone-specific antibodies were more pronounced in the low responder C57BL/6 mice strain.

Postexercise hypotension is mediated by reductions in sympathetic nerve activity.

Mean arterial pressure (MAP), the product of cardiac output (CO) and total peripheral resistance (TPR), is reduced below preexercise levels after a single bout of mild to moderate dynamic exercise. Thus acute, dynamic exercise may be used as a safe, therapeutic approach to reduce MAP. However, the mechanisms responsible for the postexercise hypotension (PEH) are unknown. We tested the hypothesis that PEH is associated with reductions in TPR and sympathetic nerve activity (SNA). Two experimental protocols were designed to test this hypothesis in male spontaneously hypertensive rats (SHR). In protocol 1 (n = 9), CO and TPR were determined before, during, and after exercise. In protocol 2 (n = 7), lumbar SNA (LSNA) was recorded before and after exercise. Rats in protocol 1 were chronically instrumented with left carotid arterial catheters and ascending aortic Doppler ultrasonic flow probes. Rats in protocol 2 were chronically instrumented with left carotid arterial catheters and electrodes around the lumbar sympathetic trunk. Dynamic treadmill exercise (9-12 m/min, 10% grade for 40 min) resulted in a postexercise reduction in MAP (from 143 +/- 5 to 128 +/- 4 mmHg, P < 0.05). Associated with the PEH was a reduction in TPR (from 28 +/- 3 to 19 +/- 2 mmHg/kHz; P < 0.05) and an elevation in CO (from 5.7 +/- 0.4 to 7.2 +/- 0.5 kHz; P < 0.05). The reductions in arterial pressure and TPR were associated with a decrease in LSNA (from 98 +/- 3 to 49 +/- 6%; P < 0.05). These results suggest that PEH is mediated by reductions in TPR and SNA.

Regulation of synthesis of complement protein C4 in human fibroblasts: cell- and gene-specific effects of cytokines and lipopolysaccharide.

Synthesis and secretion of the class III major histocompatibility complex (MHC) gene product, C4, were detected in human skin fibroblasts by metabolic labelling, immunoprecipitation and SDS-PAGE analysis. Pro-C4 (approximately 185,000 MW) was present in intracellular lysates, and the mature protein was present in extracellular media, with three bands of approximately 93,000, 75,000 and 33,000 MW, corresponding to the alpha, beta and gamma chains, respectively. C4 expression was increased in a dose-dependent manner by interferon-gamma (IFN-gamma), but was unaffected by interleukin-1 beta (IL-1 beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) alone, each of which augmented the expression of factor B, C3 and other complement proteins synthesized in fibroblasts. Simultaneous incubation of fibroblasts with IFN-gamma and TNF resulted in a synergistic increase in C4 synthesis. RNA blot analyses indicated that regulation of C4 synthesis by IFN-gamma and the combination of IFN-gamma and TNF was mediated primarily at a pretranslational level. Lipopolysaccharide (LPS) had no effect on C4 or HLA-DR synthesis in fibroblasts, either constitutive or IFN-gamma-regulated. These results are in contrast to the effects of LPS in monocytes, where LPS decreased constitutive synthesis and counter-regulated the IFN-gamma-enhanced expression of both C4 and HLA-DR. C2 expression in fibroblasts was also increased primarily by IFN-gamma. However, C2 synthesis was increased by LPS, 1L-1 and TNF, although to a lesser extent than the increase in synthesis of factor B stimulated by these mediators. These results show that up-regulation by IFN-gamma is a common feature of C2 and C4 expression in human cells that constitutively synthesize these proteins. In contrast, regulation of MHC class III and class II genes by LPS, TNF, IL-1, and IL-6 is cell- and gene-specific.

The influence of new thymopoietin derivatives on the immune response of inbred mice.

The immunomodulatory activities of new synthetic thymopoietin derivates TP4 (Arg-Lys-Asp-Val) and TP3 (Arg-Lys-Asp) have been compared to those of TP5 (Arg-Lys-Asp-Val-Tyr) which exhibits most of the biological activity of the native hormone and probably represents the active site. Both TP4 and TP3 are shown to exert similar immunomodulatory activities to TP5 affecting both humoral and cellular responses. Primary and secondary antibody responses of high responder mice were enhanced whilst the intensity of DTH reactivity was decreased. The effect on humoral immunity was particularly apparent following administration of TP4 or TP3 to mice undergoing primary antibody responses following immunization with sub-optimal doses of antigen or suppression by CY treatment. Administration of peptide(s) elicited DTH responses in mice previously shown to exhibit genetically determined unresponsiveness: in these animals antibody responses were not modulated. The data may be interpreted that the tetra- and tri-peptide representing the N-terminal sequence of TP5 possess immunomodulatory activity which is in many aspects similar to that of TP5. The experimental systems and protocols employed are shown to be appropriate for investigating the effect(s) of potential immunomodulators on humoral and cellular immunity.

Production of IgG-specific auto-anti-allotypes in b4.2-suppressed rabbits.

Rabbits were completely suppressed for kappa chain allotype b4.2, and autoantibodies against b4.1 or b4.2 could be raised in 2 out of 3 animals. The animal immunized with b4.1 produced anti-b4 and anti-Ms3, two activities which have never as yet been found together in one antiserum. Both autoantisera lacked the capacity to bind b4.2-IgM, whereas they precipitated 4.2-IgG very well. In one animal, anti-b4 IgM activity appeared after the sixth immunization, at the age of 14 months. Allotype suppression was maintained in both rabbits till the end of their lives whereas all control animals recovered within 6 months. The autoantisera which were specific for b4 IgG could not induce suppression in vivo. However, specific inhibition of b4 IgG secretion was observed in vitro.

Classes and subclasses of rat antibodies: reaction with the antigen and interaction of the complex with the complement system.

Antibodies against the dinitroaminophenyl (DNAP) haptenic group were raised in outbred CFY rats using HSA or LPS as carrier. Antibodies isolated by immunoadsorbent techniques were resolved into fractions representing distinct isotypes, and the resulting fractions were tested for avidity. Subclass IgG2a was found to contain antibodies of an avidity index lower than those of other IgG subclasses or IgM. IgG2a was the only isotype detected when DNAP-LPS was used for immunization. Complexes containing defined isotypes were compared for their capacity to activate homologous complement. IgGl type antibody-containing complexes displayed a low complement activating capacity compared with those containing IgG2b, IgG2c or IgG2a. The latter subclass when complexed with antigen can thus induce complement-dependent processes in spite of a low avidity. Insoluble complexes of IgG antibodies were rapidly solubilized in rat serum (CRA phenomenon), except those containing predominantly IgG2c.

Non-covalently bound C3 enhances lysis of rabbit erythrocytes through the alternative pathway.

Rabbit red blood cells (RaRBC, 3 x 10(7)/ml PBS) were incubated with different amounts of purified human C3 at 37 degrees for 30 min and washed twice in PBS. Different amounts of normal human serum containing 2 mM Mg2+ and 5 mM EGTA were added to the C3-treated and control RaRBC. The extent of lysis was measured after a further incubation at 37 degrees for 40 min. Enhanced lysis was observed with C3-treated RaRBC as compared to control cells. The enhancing effect was dependent on the dose of C3 used for the treatment of RaRBC. Investigation the kinetics of lysis, the lag phase was observed to be significantly shorter with the C3-treated than with the control RaRBC. No enhancement was found when RaRBC were pretreated with preformed C3b fragment. KSCN-treated C3 (C3b-like C3), however, had a lysis-enhancing effect. These results suggest that noncovalently bound C3 molecules may have a role in the initiation and/or maintenance of the alternative pathway activation on activator cells.

An inquiry-based teaching tool for understanding arterial blood pressure regulation and cardiovascular function.

Educators are placing a greater emphasis on the development of cooperative laboratory experiences that supplement the traditional lecture format. The new laboratory materials should encourage active learning, problem-solving, and inquiry-based approaches. To address these goals, we developed a laboratory exercise designed to introduce students to the hemodynamic variables (heart rate, stroke volume, total peripheral resistance, and compliance) that alter arterial pressure. For this experience, students are presented with "unknown" chart recordings illustrating pulsatile arterial pressure before and in response to several interventions. Students must analyze and interpret these unknown recordings and match each recording with the appropriate intervention. These active learning procedures help students understand and apply basic science concepts in a challenging and interactive format. Furthermore, laboratory experiences may enhance the students' level of understanding and ability to synthesize and apply information. In conducting this exercise, students are introduced to the joys and excitement of inquiry-based learning through experimentation.

Type II human complement C2 deficiency. Allele-specific amino acid substitutions (Ser189 --> Phe; Gly444 --> Arg) cause impaired C2 secretion.

Type II complement protein C2 deficiency is characterized by a selective block in C2 secretion. The Type II C2 null allele (C2Q0) is linked to two major histocompatibility haplotypes (MHC) that differ from the MHC of the more common Type I C2 deficiency. To determine the molecular basis of Type II deficiency the two Type II C2Q0 genes were isolated and transfected separately into L-cells. Subsequent molecular biology, biosynthetic, and immunofluorescence studies demonstrated that C2 secretion is impaired in Type II C2 deficiency because of different missense mutations at highly conserved residues in each of the C2Q0 alleles. One is in exon 5 (nucleotide C566 --> T; Ser189 --> Phe) of the C2Q0 gene linked to the MHC haplotype A11,B35,DRw1,BFS, C4A0B1. The other is in exon 11 (G1930 --> A; Gly444 --> Arg) of the C2Q0 gene linked to the MHC haplotype A2,B5, DRw4,BFS,C4A3B1. Each mutant C2 gene product is retained early in the secretory pathway. These mutants provide models for elucidating the C2 secretory pathway.