Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo

A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.     

Fluorescent antibody study of diseased, end-stage human kidneys.

Forty cases of diseased kidneys at end-stage were studied by fluorescent antibody technique in search for viral etiology of glomerulonephritis and other renal diseases. Among these 40 cases, 12 (30%) were ascribed to immune complex disease because of detection of immunoglobulins and complement in glomeruli of the same kidney specimen. In 8 cases (20%) only complement was detected in glomeruli. In the remaining 50% neither complement nor immunoglobulin deposit was found in glomeruli. The etiologies of the latter cases remain unknown. Of 12 cases of kidney disease of immune complex origin, hepatitis virus type B surface antigen was detected in 2 cases. In these 2 cases the magnitude of immune complex deposits with complement was greater than that of other cases. Other than hepatitis B virus antigen, no other viruses including Coxsackieviruses, ECHO viruses, and HSV-1 could be detected by indirect fluorescent antibody techniques. The proportion of complement deposit to the deposition of complement with immune complex in the diseased kidneys at end-stage was calculated and statistically analyzed.     

Actinomycin D inhibition of monoclonal antibody binding to nucleolar phosphoprotein 37/5.2 (B23)

Actinomycin D was found to block binding of a monoclonal antibody to nucleolar phosphoprotein 37/5.2 (B23) when incubated either simultaneously with or prior to addition of the antibody. Daunorubicin had no significant blocking activity of this antigen-antibody reaction. In comparative studies with a monoclonal antibody to nucleolar phosphoprotein 110/5.2 (C23), actinomycin D exhibited little blocking activity. When a 42-mer peptide containing the epitope of protein 37/5.2 (B23) was tested as the antigen, similar inhibition by actinomycin D of binding of the monoclonal antibody was found. These results provide evidence for binding of actinomycin D to a specific epitope of protein 37/5.2 (B23).     

Peptidolytic monoclonal antibody elicited by a neuropeptide.

We report evidence that a monoclonal antibody raised by immunization with a vasoactive intestinal peptide (VIP)-carrier protein conjugate selectively hydrolyzes VIP and a fluorescence quenched decapeptide (FQ14-22D), representing the region of VIP most susceptible to autoantibody-mediated cleavage (residues 14-22). A high affinity of the antibody for VIP and a lower affinity for FQ14-22D were revealed by kinetic studies and further substantiated by potent inhibition of FQ14-22D cleaving activity by full-length VIP. Sequencing of FQ14-22D hydrolysis products indicated selective cleavage at one peptide bond. These observations suggest that antibodies induced against naturally occurring polypeptide antigens can express peptidolytic activity targeted for specific sequences in the recognition epitope.     

Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.     

Protection from Streptococcus pneumoniae infection by C-reactive protein and natural antibody requires complement but not Fc gamma receptors

Streptococcus pneumoniae is an important human pathogen and the most common cause of community-acquired pneumonia. Both adaptive and innate immune mechanisms provide protection from infection. Innate immunity to S. pneumoniae in mice is mediated by naturally occurring anti-phosphocholine (PC) Abs and complement. The human acute-phase reactant C-reactive protein (CRP) also protects mice from lethal S. pneumoniae infection. CRP and anti-PC Ab share the ability to bind to PC on the cell wall C-polysaccharide of S. pneumoniae and to activate complement. CRP and IgG anti-PC also bind to Fc gamma R. In this study, Fc gamma R- and complement-deficient mice were used to compare the mechanisms of protection conferred by CRP and anti-PC Ab. Injection of CRP protected wild-type, FcR gamma-chain-, Fc gamma RIIb-, and Fc gamma RIII-deficient mice from infection. Complement was required for the protective effect of CRP as cobra venom factor treatment eliminated the effect of CRP in both gamma-chain-deficient and wild-type mice, and CRP failed to protect C3- or C4-deficient mice from infection. Unexpectedly, gamma-chain-deficient mice were extremely sensitive to pneumococcal infection. This sensitivity was associated with low levels of natural anti-PC Ab. Gamma-chain-deficient mice immunized with nonencapsulated S. pneumoniae produced both IgM- and IgG PC-specific Abs, were protected from infection, and were able to clear the bacteria from the bloodstream. The protection provided by immunization was eliminated by complement depletion. The results show that in this model of systemic infection with highly virulent S. pneumoniae, protection from lethality by CRP and anti-PC Abs requires complement, but not Fc gamma R.     

Lysis of tumor cells by antibody and complement. VII. Complement-dependent 86Rb release--a nonlethal event?

The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoidobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complete sequence of complement components. These observations indicate that 86Rb release is not a relible indicator cytotoxicity.     

Immune response in subacute sclerosing panencephalitis: reduced antibody response to the matrix protein of measles virus.

Immune precipitation was used to study the humoral immune response of patients with subacute sclerosing panencephalitis (SSPE). Patients with SSPE have a progressive infection of the CNS by measles or a measles variant despite high serum antibody levels to measles virus as measured by standard serologic techniques. However, when the antibody response to individual measles virus proteins was measured, we found a striking reduction in the ability of sera from patients with SSPE to precipitate the matrix (M) protein as compared to the precipitation of the M protein by sera from normal adults who had natural measles infection in childhood, or by convalescent sera obtained 3 to 5 weeks after a naturally occurring measles infection. The decreased antibody response to the M protein in sera from patients with SSPE occurred despite a vigorous antibody response to the other viral proteins, suggesting a selective defect in the production of antibody to a single viral protein. The reduced anti-M antibody in sera from patients with SSPE was demonstrated whether immune precipitation was performed with wild-type measles virus or SSPE virus proteins. These results suggest that in SSPE only small amounts of the M protein are produced. This result may help explain how measles virus persists in the central nervous system of patients with SSPE.     

Effect of Immunosuppressive Agents on Normal Phage-neutralizing Antibody in the Mouse

The actions of three immunosuppressive drugs on normal antibody synthesis in the adult mouse were determined. Inbred mice were given daily intraperitoneal injections of actinomycin D, uracil mustard, or cyclophosphamide for extended periods. The sera of treated and untreated mice were assayed for phage-neutralizing activity to monitor the effect of each drug on the amount of circulating normal antibody. Except for an initial decrease in titer of normal anti-phage MSP2 activity, actinomycin D had no significant effect on normal antibody activity. Uracil mustard caused alternating elevation and depression of normal antibody titers. Cyclophosphamide caused a prolonged depression of normal antibody. The response patterns to the three immunosuppressive agents were the same for both induced and normal antibody.     

Nucleotide sequence of a human monoclonal anti-idiotypic antibody specific for a rabies virus-neutralizing monoclonal idiotypic antibody reveals extensive somatic variability suggestive of an antigen-driven immune response

We previously described the isolation and characterization of a human monoclonal anti-idiotypic antibody (Ab2) isolated from EBV-transformed human PBL after immunization with rabies vaccine. The present study concerns the molecular characteristics of the Ab2 and the germ-line elements that gave rise to it. The H chain of this antibody derives from the small VHV family of human V region gene segments. Parallel studies on the germ-line VHV gene isolated from the same individual revealed that the expressed molecule contains 19 nucleotide differences in the VH gene segment. The D segment of Ab2 could have arisen by a D to D fusion; the J segment is a JH6. Extensive somatic variation evident in the H chain variable region of this naturally arising monoclonal anti-idiotypic antibody suggests that this Ab2, the product of a CD5+ B cells, was the consequence of an Ag-driven immune response.     

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.     

Control of actinomycin D biosynthesis in Streptomyces parvullus: regulation of tryptophan oxygenase activity

Tryptophan oxygenase (tryptophan 2,3-dioxygenase) activity increases immediately before the initiation of actinomycin D production by Streptomyces parvullus. We have attempted to discern whether this increase is due to a release from catabolite repression or to the synthesis of an inducer substance. The standard culture medium (glutamic acid-histidine-fructose medium) used in antibiotic production studies with S. parvullus contains l-glutamate as a major constituent. l-Glutamate is almost totally consumed before the onset of actinomycin D synthesis. The addition of 10 mM l-glutamate at this stage completely abolished actinomycin D production as well as tryptophan oxygenase synthesis. Fourteen amino acids were tested for a similar effect. Of these, l-glutamate and l-aspartate had the most dramatic effect on tryptophan oxygenase and beta-galactosidase (beta-d-galactosidase), another inducible enzyme. Standard glutamic acid-histidine-fructose medium, preincubated for 23 h to remove l-glutamate, allowed the synthesis of actinomycin D and tryptophan oxygenase by cells at a stage of growth normally considered too early for antibiotic production. A chemically defined medium lacking l-glutamate and adjusted to pH 8.0 was designed to simulate the preincubation medium. The transfer of cells to this artificial preincubation medium resulted in the appearance of tryptophan oxygenase as early as 19 h before normal synthesis occurred, eliminating the possibility that an inducer molecule is synthesized and excreted during the preincubation period. The results of these studies suggest that the increase in tryptophan oxygenase activity before the onset of actinomycin D synthesis, as well as the synthesis of actinomycin D itself, is due to a release from l-glutamate catabolite repression.     

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.     

Studies on calciferol metabolism. VII. The effects of actinomycin D and cycloheximide on the metabolism, tissue and subcellular localization, and action of vitamin D3

It was originally postulated, primarily on the basis of experiments employing actinomycin D, that calciferol (vitamin D) mediated its characteristic physiological responses in the intestine via the activation of information stored in the intestinal genome. A more recent alternative hypothesis suggested that actinomycin D blocked the biological response to calciferol by inhibiting the mandatory metabolism of cholecalciferol to 1,25-dihydroxycholecalciferol. Presented in this paper are the results of recent experiments studying the effects of both actinomycin D and cycloheximide on the metabolism, subcellular localization, and action of cholecalciferol or its metabolites, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Actinomycin D was found to inhibit calcium transport stimulated by cholecalciferol or its metabolites without inhibiting their metabolism or localization in the target tissue, the intestinal mucosa. However, actinomycin D had to be administered in four doses at 2-hr intervals to block the stimulation of calcium transport by 1,25-dihydroxycholecalciferol. Actinomycin D was also found not to lower the renal levels of 25-hydroxycholecalciferol-1-hydroxylase, which were measured in vitro. In contrast, cycloheximide was found to inhibit the localization of the sterols in the intestine. Also cycloheximide lowered the renal enzyme levels which were measured in vitro following administration of the antibiotic in vivo. From these data it can be calculated that the 25-hydroxycholecalciferol-1-hydroxylase appears to have a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of approximately 3 hr. Thus, the inhibition of intestinal calcium transport by these two antibiotics may in fact occur at two different target organs; cycloheximide by a lowering of the kidney levels of 25-hydroxycholecalciferol-1-hydroxylase and actinomycin D by blocking the action of 1,25-dihydroxycholecalciferol in the intestine.     

Rapid immune adherence reactivity of nascent, soluble antibody/DNA immune complexes in the circulation

Immune complex disease in humans and experimental animals can occur as a consequence of the binding of specific antibodies to exogenous or endogenous antigens. If this reaction occurs in the circulation, the fate of the resulting immune complex may depend upon many factors including the ability of the immune complex to fix complement and bind to complement receptors on circulating cells (immune adherence). We studied the in vivo formation and immune adherence of soluble antibody/dsDNA immune complexes in the circulation of both a nonprimate and a primate model. The fact that this sequence of biological recognition reactions is completed in less than 2 min suggests that the immune adherence phenomenon may play a crucial role in the clearance of nascent complement-fixing immune complexes from the circulation.     

Histochemical demonstration of mycobacterial antigen, specific antibody and complement in the lesions of tuberculosis

Summary Lesions were studied histochemically for mycobacterial antigen, its specific antibody and complement in 31 patients with recently-diagnosed tuberculosis. The results were related to a histological spectrum that correlated with bacterial load. The form, localization and persistence of antigen were found to be as significant as the amount. In high-resistant cases, the antigen was mainly soluble, a form which was non-toxic when ingested by macrophages but associated with tissue damage when bound to connective tissue. There was no close contact between plasma cells and antigen. However, in cases with moderate resistance, where plasma cells and antigen intermingled freely, necrosis with karyorrhexis and polymorph infiltration was associated with deposition of antigen, antibody and complement at the same sites, indicating the probability of immune complex formation in these lesions. In low-resistant cases, extensive necrosis was attributed partly to high levels of extracellular antigen.The correlation between immunological circumstances and the manifold forms of necrosis validated these forms as the basis for a histological spectrum in tuberculosis.     

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.     

Studies of the influence of different cross-linking reagents on the immune response against a B-epitope.

We have previously shown that the carrier polytuftsin obtained by polycondensation of tuftsin, a naturally occurring macrophage activator, increases significantly the antibody response against a linked B-epitope. In the present work, we have studied the influence of different cross-linking reagents on the quality of the conjugation and on the immune response, at both B-cell and T-cell levels. We observed that the cross-linking method used for coupling the B-epitope to the carrier influences the immune response. A hypothesis is put forward to explain the differences observed.     

Dirofilaria immitis: diethylcarbamazine-induced anaphylactoid reactions in infected dogs

The immunopathogenesis of the anaphylactoid Mazzotti reactions has been studied by comparing physiologic and immunologic aspects of diethylcarbamazine-induced shock in Dirofilaria immitis infected dogs with antigen induced anaphylaxis in infected and uninfected controls. Filarial antigen, specific host IgG antibody, and C1 and C3 complement levels were quantitatively measured over time in relation to the levels of histamine and prostaglandin D2 in the blood and changes in mean blood pressure. D. immitis antigen injected into uninfected dogs having no detectable IgG antibody to D. immitis or Toxocara canis produced a rapid drop in blood pressure that paralleled a drop in C1 and C3 levels and an increase in prostaglandin D2. Antigen injected into infected dogs with IgG antibody produced a similar drop in blood pressure and complement and increase in prostaglandin D2 which differed from the uninfected group only in the slower clearance of antigen from the blood. Diethylcarbamazine alone produced no measurable changes in blood pressure or complement in uninfected hosts. Diethylcarbamazine, however, administered into skin test positive infected dogs, produced a temporally slower but quantitatively similar loss in blood pressure, drop in complement, and increase in prostaglandin D2 and histamine to that induced by antigen injection. Complement activation and immune complex formation are initiated by antigen release, and subsequent vasoactive mediator release leads to shock with prostaglandin D2 being quantitatively higher in blood than is histamine.     

Strain variations in anti-sperm antibody responses and anti-fertility effects in inbred mice

This study provides evidence for polygenic controls of antisperm antibody levels in inbred male mice immunized with syngenic testis and epididymis. H2-linked and non-H2-linked genes were involved. Mice of H-2d haplotype were high responders, whereas those with H-2k haplotype were nonresponders; however, B10.D2/nSnJ mice (H-2d) were also nonresponders. In vitro fertilization inhibition by antisera correlated positively with the serum antisperm antibody levels, particularly with antibody of the immunoglobulin (Ig) G class. Inheritance of antibody response that inhibited in vitro fertilization (IVF) was an autosomal dominant trait, but this was not apparent for the control of antibody levels per se. Since IVF was inhibited by both IgG and fragment antigen-binding (Fab) isolated from immune sera, but not by immune IgG previously absorbed by sperm or testis, the biologic effect is antigen-specific and probably involved blockade of functional antigenic epitopes. Antisera to testis, caput sperm or cauda sperm were found to inhibit IVF to a similar degree. Inbred strains of mice that produced the highest levels of serum antisperm antibodies that inhibited IVF were A/J, SJL/J, DBA/1J and BALB/cByJ mice, and their antisera immunoprecipitated a common sperm antigen molecule of 35,000 to 40,000 Mr. In contrast, C57BL/6 and C57BL/10 mice produced significant antibody levels that had no effect on IVF, and their sera did not react with the 35,000- to 40,000-Mr peak. Moreover, among BALB/c H-2 congenic mice, only antiserum of responder BALB/cByJ (H-2d) mice immunoprecipitated the 35,000- to 40,000 Mr peak. Thus the 35,000- to 40,000-Mr protein may be of functional significance in the fertilization process.