Acceleration of Dextransucrase Activity of Streptococcus mutans by Secretory Immunoglobulin A

The effect of immunoglobulins on the activity of dextransucrase purified from Streptococcus mutans strain HS-6 is described. When human salivary immunoglobulin A (IgA) or colostral IgA, either natured or denatured, was incubated with dextransucrase, the rate of the dextran synthesis was markedly accelerated, whereas human serum IgA or IgG neither accelerated nor inhibited the enzyme activity. The results suggest that a portion unique for secretory IgA, the secretory component, might be related to the enzyme acceleration. On the other hand, specific rabbit antiserum against the dextransucrase inhibited completely dextran synthesis by the enzyme.     

Study of human secretory immunoglobulin A. I. Obtaining monospecific antiserum to human secretory immunoglobulin A]

A method of obtaining monospecific antiserum to the human secretory IgA is described. Immunochemically pure secretory IgA (isolated from human colostrum by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200) was used for immunization of rabbits or sheep. Heterologous antibodies were removed by adsorption with commercial gamma globulin, normal serum, the serum of a patient suffering from A-myeloma with the IgA polymere and purified lactoferrin. Monospecific antiserum to the secretory IgA gave a reaction of complete immunological identity with the secretory IgA and a free secretory component.     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

Inhibition of acrosin by oviductal secretory immunoglobulin A

A major inhibitor of acrosin in rhesus monkey and rabbit oviduct fluid, isolated by isoelectrofocusing in sucrose gradients, displayed a broad peak in the acidic region of the column and was demonstrated to contain secretory IgA specific for acrosin. Its identity was established by immunodiffusion, by the removal of acrosin inhibition with antisera to IgA (α-chain), and by its correct molecular weight during ultracentrifugation. Purified human serum IgA also inhibited rabbit, rhesus monkey, and human acrosins, but neither purified human IgG nor IgM had any inhibitory effect on these acrosins. Neither oviduct fluid secretory IgA nor purified human serum IgA inhibited the activity of bovine pancreatic trypsin. The high specificity of secretory IgA for acrosin and its presence in every rabbit and rhesus monkey oviduct fluid specimen examined suggests a possible regulatory role for this antibody in reproduction.     

An important role for polymeric Ig receptor-mediated transport of IgA in protection against Streptococcus pneumoniae nasopharyngeal carriage

The importance of IgA for protection at mucosal surfaces remains unclear, and in fact, it has been reported that IgA-deficient mice have fully functional vaccine-induced immunity against several bacterial and viral pathogens. The role of respiratory Ab in preventing colonization by Streptococcus pneumoniae has now been examined using polymeric IgR knockout (pIgR(-/-)) mice, which lack the ability to actively secrete IgA into the mucosal lumen. Intranasal vaccination with a protein conjugate vaccine elicited serotype-specific anti-capsular polysaccharide Ab locally and systemically, and pIgR(-/-) mice produced levels of total serum Ab after vaccination that were similar to wild-type mice. However, pIgR(-/-) mice had approximately 5-fold more systemic IgA and 6-fold less nasal IgA Ab than wild-type mice due to defective transport into mucosal tissues. Wild-type, but not pIgR(-/-) mice were protected against infection with serotype 14 S. pneumoniae, which causes mucosal colonization but does not induce systemic inflammatory responses in mice. The relative importance of secretory IgA in host defense was further shown by the finding that intranasally vaccinated IgA gene-deficient mice were not protected from colonization. Although secretory IgA was found to be important for protection against nasal carriage, it does not appear to have a crucial role in immunity to systemic pneumococcus infection, because both vaccinated wild-type and pIgR(-/-) mice were fully protected from lethal systemic infection by serotype 3 pneumococci. The results demonstrate the critical role of secretory IgA in protection against pneumococcal nasal colonization and suggest that directed targeting to mucosal tissues will be needed for effective vaccination in humans.     

Resistance of normal serum IgA and secretory IgA to bacterial IgA proteases: evidence for the presence of enzyme-neutralizing antibodies in both serum and secretory IgA, and also in serum IgG

Normal serum IgA and secretory IgA (sIgA) of subclass IgA1 were isolated from pooled human serum and milk, respectively. They were tested for their susceptibility to bacterial IgA proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Neisseria meningitidis that cleave IgA of only the IgA1 subclass. They were also tested for susceptibility to a novel IgA-protease from Clostridium ramosum that cleaves IgA of the IgA1 as well as the IgA2 subclass of the A2m(1) allotype. Both normal serum IgA1 and sIgA1 exhibited resistance to most IgA proteases. The one exception was the IgA protease from C. ramosum which readily cleaved both the serum IgA1 and sIgA1 into Fab and Fc fragments. Secretory component (SC) had nothing to do with the resistance of these IgAs. The resistance of these IgAs to most of the IgA proteases was found to be due to their enzyme-neutralizing antibody activity, since the Fab but not the Fc fragment of sIgA1 showed enzyme-inhibitory activity against these IgA proteases. Similar enzyme-neutralizing antibody activity was found in the pepsin-digested normal serum IgG-(Fab')2 fragment. These results indicate that the induction of the enzyme-neutralizing antibodies against the bacterial IgA proteases took place not only in mucosal sIgA but also in serum IgA and IgG. No enzyme-neutralizing antibody activity against the novel IgA-protease of C. ramosum was detected in any immunoglobulin preparations used in the present study or in the serum of a patient who carries the IgA protease-producing strain of C. ramosum in his feces.     

Antimicrobial Effect of Human Serum IgA

Serum IgA, IgG and colostrum secretory IgA prepared from specimens pooled from a large number of human beings were shown to have measurable levels of antibodies against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, poliovirus, Coxsackie B virus, echovirus and influenza virus. Serum IgA exerted a bacteriostatic effect in vitro on E. coli and P. aeruginosa, which increased in the presence of the iron-binding proteins lactoferrin and transferrin. This bacteriostasis was reduced when the iron-binding proteins were saturated with iron. Similar results were obtained with IgG and secretory IgA. The bacteriostatic effect of serum IgA was also shown in vivo, in the peritoneal cavity of mice. The effect was suppressed by iron. Iron-chelating substances, siderophores, excreted by E. coli diminished the cosoperative bacteriostatic effect of serum IgA and transferrin. Siderophore production by E. coli was inhibited in the presence of serum IgA, but not when serum IgA was deprived of specific antibody by absorption with E. coli. These results indicate that serum IgA has a potent bacteriostatic effect in cooperation with transferrin or lactoferrin because of the inhibitory effect of the specific antibody on siderophore production by E. coli.     

Mannose-containing oligosaccharides of non-specific human secretory immunoglobulin A mediate inhibition of Vibrio cholerae biofilm formation

The role of antigen-specific secretory IgA (SIgA) has been studied extensively, whereas there is a limited body of evidence regarding the contribution of non-specific SIgA to innate immune defenses against invading pathogens. In this study, we evaluated the effects of non-specific SIgA against infection with Vibrio cholerae O139 strain MO10 and biofilm formation. Seven day old infant mice deficient in IgA (IgA(-/-) mice) displayed significantly greater intestinal MO10 burden at 24 hr post-challenge when compared to IgA(+/+) pups. Importantly, cross-fostering of IgA(-/-) pups with IgA(+/+) nursing dams reversed the greater susceptibility to MO10 infection, suggesting a role for non-specific SIgA in protection against the infection. Since biofilm formation is associated with virulence of MO10, we further examined the role of human non-specific SIgA on this virulence phenotype of the pathogen. Human non-specific SIgA, in a dose-dependent fashion, significantly reduced the biofilm formation by MO10 without affecting the viability of the bacterium. Such an inhibitory effect was not induced by human serum IgA, IgG, or IgM, suggesting a role for the oligosaccharide-rich secretory component (SC) of SIgA. This was supported by the demonstration that SIgA treated with endoglycosidase H, to cleave the high-mannose containing terminal chitobiose residues, did not induce a reduction in biofilm formation by MO10. Furthermore, the addition of free mannose per se, across a wide dose range, induced significant reduction in MO10 biofilm formation. Collectively, these results suggest that mannose containing oligosaccharides within human non-specific secretory IgA can alter important virulence phenotypes of Vibrio cholerae such as biofilm formation, without affecting viability of the microorganism. Such effects may contribute significantly to innate immune defenses against invading pathogens in vivo in the gastrointestinal tract.     

Studies on human gastric mucosal immunoglobulin A.

Immunoglobulin A (IgA) was found in mucus scraped from the surface of the human antrum. Fresh human gastric mucosa removed at operation was washed free of loosely adhering material and the gelatinous mucus lining the tissue scraped. The scrapings were separated by gel filtration on Sephadex G-200 and on Sepharose 4B into two carbohydrate-containing fractions. One of these fractions was shown by immunodiffusion to contain IgA which differs from human colostral secretory IgA by being devoid of secretory component activity. Moreover, secretory component was not detected in our unfractionated gastric mucosal scrapings. It is concluded that, contrary to the general belief, the predominant immunoglobulin A of human gastric mucus is not associated with the secretory component. Our results do not exclude the possibility that, as in serum, small amounts of secretory IgA and of the secretory component may be present in gastric secretions, however if so, the levels of these compounds would fall below the level of sensitivity of our methods.     

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.     

Subclass distribution of antigen-specific IgA antibodies in normal donors and individuals with homozygous C alpha 1 or C alpha 2 gene deletions

To analyze the subclass restriction of Ag-specific IgA, sera and saliva from healthy blood donors and from IgA class or subclass deficient individuals were studied. The latter included donors with or without C alpha 1 or C alpha 2 gene deletions. Monoclonal human IgA1 and a genetically engineered IgA2 antibody, normal human serum and colostrum IgA were used as standards to estimate serum and saliva levels of Ag-specific antibodies. In normal individuals, there was a strong IgA1 preference of naturally acquired antibodies in serum against both polysaccharide Ag (PPS 6A, PPS 23, pneumococcal C-polysaccharide, and LPS from Escherichia coli) and protein Ag (Staphylococcus aureus alpha-toxin and HSV). Specific IgA2 in serum against the tested Ag were frequently not measurable. In contrast, most of the individuals with homozygous C alpha 1 gene deletions displayed substantial amounts of specific IgA2 against protein as well as polysaccharide Ag. The median levels of specific IgA in serum against protein Ag were approximately one-third as compared to normal individuals and one-fifth, or less, against polysaccharide Ag. Normal serum levels of IgA against the tested Ag, restricted to the IgA1 subclass, were noted in two individuals with IgA2 deficiency, one of whom carried a homozygous C alpha 2 gene deletion. Median values of specific IgA, against the tested Ag S. aureus alpha-toxin, HSV, and pneumococcal C-poly-saccharide, from normal healthy donors were approximately four to eight times higher in serum as compared to saliva. Individuals with homozygous C alpha 1 gene deletions displayed increased levels of the various specific IgA2 antibodies in saliva. In conclusion, the individuals with homozygous C alpha 1 gene deletions displayed decreased median levels of specific IgA antibodies in serum despite normal levels of total IgA. Normal levels of both specific IgA and total IgA in saliva were found.     

Human IgA inhibits adherence of Acanthamoeba polyphaga to epithelial cells and contact lenses

Specific anti-Acanthamoeba IgA antibodies have been detected in the serum and tears of patients and healthy individuals. However, the role of human secretory IgA antibodies in inhibiting the adherence of Acanthamoeba had not been previously investigated. Therefore, the purpose of this study was to purify secretory IgA from human colostrum and analyze its effect on the adherence of Acanthamoeba trophozoites to contact lenses and Madin-Darby canine kidney (MDCK) cells. IgA antibodies to Acanthamoeba polyphaga in colostrum of healthy women as well as in saliva and serum of healthy subjects were analyzed by ELISA and Western blot analysis. In serum, saliva, and colostrum, we detected IgA antibodies that recognized several antigens of A. polyphaga. In addition, colostrum and IgA antibodies purified from it inhibited adherence of A. polyphaga trophozoites to contact lenses and MDCK cells. These results suggest that IgA antibodies may participate in the resistance to the amoebic infection, probably by inhibiting the adherence of the trophozoites to contact lenses and corneal epithelial cells.     

Immunization against influenza in humans using an oral enteric-coated killed virus vaccine

By ingestion of subunit-killed influenza virus vaccine in the form of enteric-coated capsules, local synthesis of secretory IgA (sIgA) antibody was stimulated in human nasal secretions. A fairly equal antibody response initiated by oral and intramuscular administration was demonstrated in the nasal secretions, although a systemic immune response was not elicited from ingestion of the vaccine. If the secretory antibody response resulted from absorption of antigen and transport to the respiratory mucosa, systemic (serum) antibody would be expected. Therefore these findings support the hypothesis that specialized collections of lymphoid cells in the small intestines have IgA precursor cells which circulate and populate distant mucosal sites. A number of studies have suggested that protection against mucosal infection by a variety of respiratory viruses correlates better with the presence and level of sIgA antibody than with serum antibody. The orally administered vaccine was associated with no more side effects than placebo, in contradistinction to the intramuscular route. Thus, the oral method of influenza vaccination could prove to be superior in providing for immunological protection due to equal secretory antibody stimulation, improved convenience and less toxicity.     

Activity of human IgG and IgA subclasses in immune defense against Neisseria meningitidis serogroup B

Both IgG and IgA Abs have been implicated in host defense against bacterial infections, although their relative contributions remain unclear. We generated a unique panel of human chimeric Abs of all human IgG and IgA subclasses with identical V genes against porin A, a major subcapsular protein Ag of Neisseria meningitidis and a vaccine candidate. Chimeric Abs were produced in baby hamster kidney cells, and IgA-producing clones were cotransfected with human J chain and/or human secretory component. Although IgG (isotypes IgG1-3) mediated efficient complement-dependent lysis, IgA was unable to. However, IgA proved equally active to IgG in stimulating polymorphonuclear leukocyte respiratory burst. Remarkably, although porin-specific monomeric, dimeric, and polymeric IgA triggered efficient phagocytosis, secretory IgA did not. These studies reveal unique and nonoverlapping roles for IgG and IgA Abs in defense against meningococcal infections.     

The specificity of the IgA receptor purified from human neutrophils.

A receptor for IgA was purified from human polymorphonuclear neutrophils (PMN) by affinity chromatography on human serum IgA-Sepharose. The receptor appeared on SDS/polyacrylamide gels as a diffuse band with an apparent molecular mass of 50-70 kDa, whether reduced or non-reduced. During purification, the protein showed remarkable stability to proteolytic digestion by endogenous PMN proteinases. Purified radioiodinated receptor re-bound to IgA-Sepharose, but not to IgG-Sepharose or BSA-Sepharose. The binding of the receptor to IgA-Sepharose was inhibited in a dose-dependent manner by human serum IgA1 or IgA2 or secretory IgA1 or IgA2, but not by IgG or IgM. Binding of receptor to IgA-Sepharose was also inhibited by the Fc fragment of IgA, but not by the Fab fragment. An IgA fragment produced by digestion with pepsin which lacks the CH3 domain also inhibited binding, but to a more limited extent than did the whole IgA molecule.     

A novel receptor for secretory component on porcine mononuclear cells

Porcine mononuclear cells from peripheral blood separated by Ficoll-Hypaque gradient technique bound secretory IgA preferentially but not serum IgA or IgG after capping with anti-Ig. When the cells were incubated with free secretory component and washed, serum IgA binding was facilitated. Thus, the results indicate that a subpopulation of porcine circulating mononuclear cells bear surface receptors for secretory component (SC-receptor).     

Lack of correlation between serum and salivary concentration levels of immunoglobulin A and lysozyme (muramidase)

The study was conducted in a group of 232 individuals comprising 114 males and 118 females 20 to 67 years old. As revealed by the linear regression analysis method, mean serum concentration levels of immunoglobulin A (IgA) and lysozyme (LYS) were unrelated to values found in the spontaneously secreted saliva specimens. Levels of serum IgA were clearly age-dependent, but no such correlation was recorded in the case of secretory IgA. These findings suggest that tests for salivary IgA can hardly be taken as an equivalent to IgA determinations in the human serum.     

Kinetics of the mucosal antibody secreting cell response and evidence of specific lymphocyte migration to the lung after oral immunisation with attenuated S. enterica var. typhimurium

The kinetic of mucosal secretory responses elicited by the vaccine vector Salmonella enterica var. typhimurium (S. typhimurium) was examined by enzyme linked immunospot (ELISPOT) and compared with serum responses. Mice immunised orally with BRD509, the aroA, aroD mutant of virulent S. typhimurium SL1344 expressing the C Fragment of tetanus toxin (TT), simultaneously developed an IgA antibody secreting cells (ASC) response in the gastrointestinal lamina propria, the spleen and the lung, against both S. typhimurium lipopolysaccharide (LPS) and TT. The magnitude of the ASC response was greatest in the gut, was boosted by a secondary immunisation at day 25, and the kinetic of the response did not correlate with the appearance of serum antibodies. This study suggests that S. typhimurium can engage the common mucosal immune system to effect mucosal secretory responses at distal sites, however, the magnitude of the responses is both greatest in the gut and antigen-specific. The ASC origin of the serum antibodies specific for S. typhimurium and antigens expressed by the bacterium is yet to be elucidated.     

Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity

The protozoan Entamoeba histolytica is the etiological agent of amebiasis, an infection with high prevalence worldwide. The host-ameba relationship outcome depends on parasite and host factors, and among these is secretory IgA. These antibodies reduce mucosal colonization by pathogens and neutralize a variety of toxins and enzymes. The functionality of secretory IgA depends on its integrity. Some bacteria produce IgA proteases that cleave mainly the IgA1 subclass; live E. histolytica trophozoites, and other ameba fractions are also able to degrade human IgA. The aim of this study was to determine if serum and secretory IgA, its subclasses and secretory component, are degraded by cysteine proteases, which are present and active on the surface of glutaraldehyde-fixed amebas. It was observed that secretory IgA1, IgA2, free and IgA-bound secretory component were degraded by E. histolytica surface-associated cysteine proteinases. Secretory IgA2, although it was degraded, conserved its ability to agglutinate live amebas better than IgA1. Therefore, while specificity of known ameba cysteine proteases is cathepsin B-like and is different from bacterial IgA proteases, IgA2 was functionally more resistant than IgA1 to ameba surface-associated cysteine protease degradation, similar to the greater resistance of IgA2 to bacterial IgA-specific proteases.     

Mac-1 (CD11b/CD18) as accessory molecule for Fc alpha R (CD89) binding of IgA

IgA, the principal ligand for FcalphaRI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human FcalphaRI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the beta(2) integrin Mac-1 (Mac-1(-/-)) were unable to bind SIgA, despite expression of FcalphaRI. Consistent with this, serum IgA stimulated Mac-1(-/-) PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1(-/-) PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-FcalphaRI interactions.