Human Secretory IgM Antibodies Activate Human Complement and Offer Protection at Mucosal Surface.

IgM molecules circulate in serum as large polymers, mainly pentamers, which can be transported by the poly-Ig receptor (pIgR) across epithelial cells to mucosal surfaces and released as secretory IgM (SIgM). The mucosal SIgM molecules have non-covalently attached secretory component (SC), which is the extracellular part of pIgR which is cleaved from the epithelial cell membrane. Serum IgM antibodies do not contain SC and have previously been shown to make a conformational change from 'a star' to a 'staple' conformation upon reaction with antigens on a cell surface, enabling them to activate complement. However, it is not clear whether SIgM similarly can induce complement activation. To clarify this issue, we constructed recombinant chimeric (mouse/human) IgM antibodies against hapten 5-iodo-4-hydroxy-3-nitro-phenacetyl (NIP) and in addition studied polyclonal IgM formed after immunization with a meningococcal group B vaccine. The monoclonal and polyclonal IgM molecules were purified by affinity chromatography on a column containing human SC in order to isolate joining-chain (J-chain) containing IgM, followed by addition of excess amounts of soluble SC to create SIgM (IgM J+ SC+). These SIgM preparations were tested for complement activation ability and shown to be nearly as active as the parental IgM J+ molecules. Thus, SIgM may offer protection against pathogens at mucosal surface by complement-mediated cell lysis or by phagocytosis mediated by complement receptors present on effector cells on mucosa.

Different glycosylation pattern of human IgG1 and IgG3 antibodies isolated from transiently as well as permanently transfected cell lines.

The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cell-mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.

Pectic polysaccharides isolated from Malian medicinal plants protect against Streptococcus pneumoniae in a mouse pneumococcal infection model.

The aim of this study was to investigate whether the pectic polysaccharides BP-II, Oc50A1.I.A and CC1P1 isolated from the Malian medicinal plants Biophytum petersianum, Opilia celtidifolia and Cola cordifolia, respectively, were able to protect against Streptococcus pneumoniae infection in mice. The pectin preparations were administered intraperitoneally 3 h before challenge with S. pneumoniae serotype 6B. Blood samples were obtained from all animals before and at 3 h, 24 h and 72 h after challenge with the pneumococci. The number of bacteria in blood was recorded and the blood concentration of a range of cytokines measured. The pretreatment with BP-II, Oc50A1.I.A and CC1P1 demonstrated a protective activity against S. pneumoniae serotype 6B infection, albeit at different range of concentrations. The pectins showed no direct antibacterial effects towards S. pneumonia; however, they induced the production of a range of cytokines and chemokines. We have previously shown that BP-II, Oc50A1.I.A and CC1P1 exhibit complement fixation activity and also that BP-II and Oc50A1.I.A stimulate macrophages to produce NO. The observed clinical effect might therefore be linked to the ability of the pectic polysaccharides to stimulate the innate immune system.

Similar superantigen gene profiles and superantigen activity in norwegian isolates of invasive and non-invasive group a streptococci.

Group A streptococcus (GAS) harbours several virulence factors, including M protein (coded by the emm gene) and superantigens (SAgs). SAgs are extracellular toxins that directly activate the immune system by cross-binding to the HLA class II molecule and T cell receptor (TCR), thereby causing activation of up to 30% of the T cells and subsequent massive secretion of cytokines. Forty-eight GAS strains isolated from patients at Norwegian hospitals between 1988 and 2004 were included in this study. Of these, 24 were invasive streptococcal toxic shock syndrome (STSS) or necrotizing fasciitis (NF) isolates and 24 were non-invasive pharyngitis isolates, matched for having the same T-type and year of isolation as the invasive isolates. The isolates were characterized by emm sequence typing, multilocus sequence typing (MLST) and SAg gene profiles. A correlation between T-type, emm type, sequence type and SAg gene profile was revealed. No difference between invasive and non-invasive isolates regarding serotype or genotype was demonstrated. Selected invasive and non-invasive isolates with identical SAg gene profiles were analysed for SAg activity in bacterial growth culture media with and without human cell culture media added. A human T cell proliferation assay was used as measurement for SAg activity and simultaneously we also measured the cytokine content in normal human peripheral blood leucocyte cell culture media. The results revealed that invasive and non-invasive isolates did not differ significantly in SAg activity as it is present in semipurified bacterial culture medium.

Evidence for recent duplications among certain gamma globulin heavy chain genes.

A survey of a large number of human sera with the heavy chain genetic markers of the gamma-globulin system has revealed an unusual gene complex which is inherited as a unit through two different families. The gene complex involves two pairs of gammaG1 genetic markers which ordinarily behave as homoalleles, Gm(z) and Gm(f) for the Fd part of gammaG1 molecules, and Gm(a) and non-a for the Fc part. Isolation of the gammaG1 fraction from the unusual sera demonstrated the presence of the important non-a antigen in the gammaG1 fraction. Through the use of immunoadsorbents it was shown that these antigens were not part of a single molecule but that separate molecules were involved. The accumulated evidence indicated that the appearance of such homoalleles on the same chromosome probably resulted from a recent gene duplication, giving rise to two gammaG1 cistrons on one chromosome.

Structural difference in the complement activation site of human IgG1 and IgG3.

The C1q binding epicentre on IgG molecules involves residues Asp(270), Lys(322), Pro(329) and Pro(331) in the C(H)2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5-iodo-4-hydroxy-3-nitro-phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody-dependent complement-mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10-fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp(270), Leu(334), Leu(335). For all these residues, and especially for Asp(270), IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.

Antibody response to long-term and high-dose mould-exposed sawmill workers.

Exposure to moulds is thought to cause adverse health effects ranging from vague subjective symptoms to allergy and respiratory diseases. Until now, most studies have been emphasizing low levels of exposure. In Norwegian sawmills during the 1980s, extensively high spore counts up to 10(7) spores/m3 air were reported. By using serum samples obtained from sawmill workers during that period, in addition to control sera, we studied the antibody response of all classes and IgG subclasses to Rhizopus microsporus at different levels of exposure. Antigen specificity was further studied by Western blotting. Exposure to R. microsporus was accompanied by R. microsporus-specific antibody production against a wide range of antigenic components most likely of both protein and carbohydrate nature. Increasing levels of mould-specific IgG1, IgG2, IgG4 and IgA antibodies were associated with increased exposure, while the highest levels of exposure were associated with a somewhat reduced level of mould-specific IgE antibodies. In conclusion, the present study strongly suggests that high mould exposure can induce a strong IgG and IgA response in a dose-dependent manner.

Characterization of an FcgammaRI-binding peptide selected by phage display.

The high-affinity IgG receptor, Fcgamma receptor I (FcgammaRI), is expressed exclusively on myeloid cells, and there is a great interest in the targeting of vaccine antigens to FcgammaRI using anti-human FcgammaRI antibodies or fragments derived from such molecules. In order to reduce the size and complexity of the targeting reagent, we have searched for FcgammaRI binding peptides in peptide libraries displayed on phage. The human monocytic cell line U937 was used as target. Phages that displayed the consensus peptide CLRSGXGC were selected and revealed increased binding to IFN-gamma stimulated versus non-stimulated U937 cells as well as to FcgammaRI transfected versus non-transfected IIA1.6 cells. Furthermore, they bound the extracellular domains of soluble FcgammaRI, but neither FcgammaRIIA, FcgammaRIIB nor FcgammaRIIIB. Binding was inhibited by a synthetic version of the peptide, whereas neither human IgG nor the FcgammaRI-specific monoclonal antibodies (mAb) mAb22 and 32.2 interfered. Flow-cytometry analysis and internalization studies showed that a synthetic biotin-conjugated peptide ADGACLRSGRGCGAAK-bio was able to target U937 cells and FcgammaRI transfected IIA1.6 cells, and further to promote internalization and vesicular degradation of streptavidin coupled to 1 microm magnetic beads. These peptides may have potential as FcgammaRI targeting reagents.

Efficient purification of DNA fragments using a protein binding membrane.

A novel and efficient method has been developed for isolation of correctly digested DNA fragments without the use of classic size-dependent electrophoretic separation methods. To achieve this, DNA fragments are end-labelled by haptens. After specific endonuclease digestion of the hapten-labelled DNA, the DNA is incubated with a protein that specifically binds to the hapten. The incubation mixture is then passed through a cartridge containing a protein-binding membrane that does not bind DNA. Undigested and partly digested DNA are retained on the membrane, while correctly digested DNA is selectively recovered for use in further downstream applications.

Human IgG1, IgG3, and IgG3 Hinge-Truncated Mutants Show Different Protection Capabilities against Meningococci Depending on the Target Antigen and Epitope Specificity.

We compared the bactericidal activity of recombinant sets of chimeric IgG monoclonal antibodies against two important outer membrane meningococcal vaccine antigens: PorA and factor H binding protein (FHbp). The sets contained human Fc portions from IgG1, IgG3, and two IgG3 mutants (IgG3m15 and IgGm17) with hinge regions of 15 and 17 amino acids encoded by hinge exons h2 and h1, respectively (human IgG3 has a hinge region of 62 amino acids encoded by hinge exons h1, h2, h3, and h4, while human IgG1 has a hinge region of only 15 amino acids encoded by one hinge exon) and mouse V regions. IgG1 showed higher bactericidal activity than IgG3 when directed against PorA (an abundant antigen), while IgG3 was more bactericidal than IgG1 when directed against FHbp (a sparsely and variably distributed antigen). On the other hand, the IgG3 hinge-truncated antibodies IgG3m15 and IgGm17 showed higher bactericidal activity than both IgG1 and IgG3 regardless of the target antigen. Thus, the Fc region of IgG3 antibodies appears to have an enhanced complement-activating function, independent of their long hinge region, compared to IgG1 antibodies. The greater activity of the truncated IgG3 hinge mutants indicates that the long hinge of IgG3 seems to downregulate through an unknown mechanism the inherent increased complement-activating capability of IgG3 Fc when the antibody binds to a sparse antigen.

Effect of streptolysin O and digitonin on egg lecithin/cholesterol vesicles.

Artificial membrane vesicles (liposomes) have been used to study the lytic mechanism of the bacterial toxin, streptolysin O, compared to that of the well-known plant glycoside, digitonin. Two types of vesicle were prepared: large unilamellar vesicles and multilamellar liposomes. The vesicles were prepared with varying molar ratios of egg lecithin and cholesterol and loaded with the water-soluble spin label, TEMPO-choline chloride. Lysis of the vesicles was registered as release of spin label and monitored by change in the electron spin resonance (ESR) spectrum. In this system digitonin was able to lyse both large unilamellar vesicles and multilamellar liposomes. The effectiveness of lysis increased by increasing the percentage of cholesterol, but even at 0% cholesterol a significant level of lysis was observed by addition of a large enough concentration of digitonin. In contrast, no lysis was detected from multilamellar liposomes after exposure to streptolysin O, even when they consisted of 50 mol% cholesterol. On the other hand, large unilamellar vesicles could be lysed by streptolysin O, provided the cholesterol content was greater than 33%. At 67 mol% cholesterol in the membranes, the degree of lysis was diminished compared to 50%, which appeared to be optimal. This is the first demonstration of liposome lysis by streptolysin O and demonstrates the cholesterol specificity which has previously been shown by inhibition studies.

Alteration of the conformation of human IgG subclasses by reduction of the hinge S-S bonds.

Human IgG changed molecular size upon mild reduction and alkylation as shown by HPLC gel filtration. IgG1, IgG2 and IgG4 proteins increased in molecular size while IgG3 proteins were decreased in molecular size by this treatment. Several proteins within each subclass covering different light chain types and Gm types were tested all showing the same effect. A plausible explanation was related to the hinge and to the CH2 region since Fab fragments experienced unchanged molecular size irrespective of IgG subclass while Fc (of IgG1, IgG2, IgG3, containing only two aa of the 62 aa long hinge of IgG3 and IgG4) increased in size and Fch (which contains most of the 62 aa long hinge region of IgG3) decreased in size upon reduction and alkylation. It is postulated that reduction of the hinge S-S bonds permit the IgG and Fc molecules to open up in the CH2 region due to the lack of trans-interaction here, resulting in a larger molecular size. For IgG3 and Fch (from IgG3) molecules there was an opposite and even greater effect on the open polyproline like structure of the gamma 3 hinge which depends on intact S-S bonds (there are 11 bonds here). Reduction of these S-S bonds apparently breaks down this open hinge structure resulting in a net decrease in molecular size of IgG3 and Fch molecules.

Human IgG3 is decreased and IgG1, IgG2 and IgG4 are unchanged in molecular size by mild reduction and reoxidation without any major change in effector functions.

Purified proteins of the four human IgG subclasses were reduced under neutral conditions to break the interchain S-S bonds, followed by dialysis to allow reformation of S-S bonds (pr/o treatment). The IgG1, IgG2 and IgG4 proteins apparently reformed native molecules by pr/o treatment, while IgG3 formed molecules with significantly smaller size, as measured by HPLC gel filtration, compared to the autologous native proteins. The degree of shrinking of the pr/o IgG3 molecules varied and was most pronounced at low protein concn. In addition, the temp and the concn of reducing agent during the pr/o treatment had some influence on the molecular size. The effect is probably due to a conformational change of the 62 amino acid long hinge of IgG3. The effector activity of pr/o IgG2 and pr/o IgG3 was studied by employing chimeric, mouse V and human C regions, monoclonal antibodies with the same NIP-binding properties. Thus, the interaction between IgG and the complement system was unchanged both for pr/o IgG2 and pr/o IgG3, while the Fc-receptor-mediated antibody-dependent cellular cytotoxicity (ADCC) was depressed to the same degree for both pr/o IgG2 and pr/o IgG3. Conclusively, the alteration of the conformation of the IgG3 molecule by pr/o treatment had no major influence on its effector functions.

Antibody dependent cell-mediated cytotoxicity induced by chimeric mouse-human IgG subclasses and IgG3 antibodies with altered hinge region.

A matched set of chimeric mouse-human NP-antibodies were studied for the capacity to induce cell mediated cytotoxicity (ADCC) by normal peripheral blood NK/K cells. The target cells were sheep red blood cells (SRBC) sensitized with the haptens NP or NIP. All four IgG subclasses and several IgG3 variants with altered hinge were tested for ADCC activity. The hierarchy of the ADCC capacity among the subclasses was found to be IgG3 greater than IgG1 greater than IgG4 greater than IgG2. The superiority of IgG3 was only revealed at low effector cell:target cell ratio. The ADCC activity was for the most part unaltered by shortening the hinge region of IgG3 from 62 to 15 amino acids. Also, when the hinge region of IgG3 was mutated to become identical to that of IgG4, the ADCC activity was mainly unchanged. However, an IgG3 variant with deletion of all four hinge exons showed a depressed ADCC activity compared to the wild type. The IgG subclass pattern of complement-mediated lysis (CML) and ADCC is different and the capacity to induce CML and ADCC is changed differently by hinge region modification. Thus CML and ADCC have different structural requirements in the Fc region of IgG.

Human IgG3 can adopt the disulfide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis.

In this paper we describe the construction of mouse-human IgG3 mutant antibodies resembling IgG1 in their disulfide bond pattern between the heavy and light chain (H-L) and between the two heavy chains (H-H). The effector functions of these mutant antibodies were compared to normal IgG3 and IgG1. Changing only the disulfide bond pattern between the heavy and light chains did not alter the ability to induce complement mediated cell lysis (CML), regardless of the amount of corresponding antigen that had been introduced to the surface of the target cells. However, alteration of the disulfide bond pattern between the two heavy chains had a large effect on CML due to shortening of the hinge from 62 to 15 amino acids. No difference between the mutants and normal antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC) was observed. This suggests that IgG3 can adopt the H-L disulfide bond pattern of IgG1 without obtaining the CML activity characteristic for IgG1.

Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation.

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

H NMR studies of the Fc region of human IgG1 and IgG3 immunoglobulins: assignment of histidine resonances in the CH3 domain and identification of IgG3 protein carrying G3m(st) allotypes.

A 1H NMR study of the Fc region of human IgG1 and IgG3 immunoglobulins is presented. 1H NMR data were collected for the Fc and pFc' fragments obtained from human monoclonal IgG1 and IgG3 and also from rabbit IgG. The C2-H proton signal of His-435 in the CH3 domain of IgG1 was assigned by comparing the spectra of the Fc fragment of IgG1 with that of IgG3 [G3m(g)] where there is a substitution of histidine by arginine at position 435. Chemical shifts and linewidths of the His-435 signal are quite different for the Fc and pFc' fragments. We suggest that His-435 is involved in the interdomain CH2-CH3 contact. Assignments of the C2-H proton signals of His-429 and His-433 in the CH3 domain were made on the basis of our previous 1H NMR results on the human light chain. NMR measurements clearly show that IgG3 Jir, which was isolated from a Japanese patient with cryoglobulinemia, has histidine at position 435 as in the case of IgG1. We also confirmed that IgG3 Jir reacts strongly with protein A. In marked contrast, IgG3 [G3m(g)] does not bind protein A. These results show that binding of protein A to the Fc region is not subclass-specific and the existence of His-435 is a necessary condition for the protein A binding. It has recently been demonstrated that IgG3 proteins carrying G3m(st) allotypes, which are relatively common in Mongoloid populations but quite rare in Caucasians, bind protein A strongly. We confirmed that, as expected, IgG3 Jir carries G3m(st) allotypes. The pH titration curve of His-435 observed for IgG3 Jir is quite different from that for IgG1. This result makes it possible to identify by 1H NMR IgG3 proteins carrying G3m(st) allotypes. In the case of the pFc' fragments, His-435 gives identical titration curves for IgG1 and IgG3 [G3m(st)]. This is consistent with the fact that no serological distinction can be made between the pFc' fragments obtained from these two types of proteins. We suggest that G3m(st)-specific antiserum differentiates IgG1 and IgG3 [G3m(st)] by recognizing the difference in the way in which the CH2 and CH3 domains make contact with each other.

The use of a hapten-Fab conjugate to sensitize target cells for antibody-dependent complement-mediated lysis and antibody-dependent cell-mediated cytotoxicity.

NIP-conjugated Fab' fragments from a rabbit hyperimmunized with sheep red blood cells (SRBC) were used to link the hapten NIP to target cells for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent complement-mediated lysis (ADCML). Target cells (SRBC) labelled with this NIP-Fab' complex were compared with SRBC directly haptenized with NIP in ADCC and ADCML assays using a NIP specific IgG1 chimeric antibody. Both methods yielded almost identical results. Using the NIP-FAb' conjugate identical target cell haptenization was readily achieved from experiment to experiment. Using conjugates of different NIP/Fab' ratios it should be possible to study how such changes influence antibody effector functions.

Human IgG subclass-specific rabbit antisera suitable for immunoprecipitation in gel, ELISA and multilayer haemagglutination techniques.

Antisera specific for human IgG subclasses were raised in rabbits by tolerance induction at birth followed by active immunization at 8 weeks of age. This procedure gave better antisera compared to tolerance/immunization of adult grown animals. Many animals gave antisera with subclass-specific precipitating activity without the need to use absorption procedures. However, for sensitive techniques such as ELISA and multilayer haemagglutination it was necessary to absorb the antisera to make them subclass-specific. Subclass specificity in ELISA and haemagglutination was verified by inhibition. We believe this is the first description of a procedure to prepare antisera specific for human IgG subclasses suitable for use in immunoprecipitation, ELISA and haemagglutination techniques.