Selective transport of polymeric immunoglobulin A in bile. Quantitative relationships of monomeric and polymeric immunoglobulin A, immunoglobulin M, and other proteins in serum, bile, and saliva.

In 17 adults, serum, hepatic bile, and saliva samples were analyzed for their sedimentation profile of IgA and secretory component (SC), and for their concentrations of albumin, orosomucoid, transferrin, IgG, IgA, alpha 2-macroglobulin (alpha 2M), IgM, and SC. Polymeric IgA(p-IgA) averaged 13% (50-700 micrograms/ml) of total IgA in serum, 70% (43-88%) in bile, and 93% (74-98%) in saliva. Most of the p-IgA in bile sedimented with SC, which also occurred free (8-44%), and with IgM. In bile, albumin (155-1,485 micrograms/ml) was the predominant protein, followed by IgG (32-480 micrograms/ml), and total IgA (37-209 micrograms/ml). In saliva, p-IgA (72-902 micrograms/ml) predominated, followed by albumin (16-385 micrograms/ml) and IgG (9-178 micrograms/ml). Secretion-to-serum albumin-relative concentration ratios (S/S-ARCR = 1 for albumin) in bile averaged 22 for p-IgA, 1.91 for IgM, 1.28 for monomeric IgA (m-IgA), 0.70 for IgG, and 0.57 for alpha 2M, indicating for p-IgA, IgM, and to a lesser extent for m-IgA, a selective excretion into bile. In saliva, a 16-fold greater selective excretion of p-IgA (mean S/S-ARCR = 354) was found. Labeled m- and p-IgA were injected intravenously into five patients. Specific activities indicated that for p-IgA 50% was serum derived in bile, as compared with 2% in saliva, and to 85% for m-IgA in bile. In the patient with the highest excretion of 125I-p-IgA in bile, only 2.8% of the injected dose was recovered in bile within 24 h after injection. Compared with rats and rabbits, the serum-to-bile transport of p-IgA in humans is much smaller.     

Participation of food antigens in increased polyclonal antibody production induced by alcohol

In order to obtain a better understanding of hyperglobulinemia in chronic alcoholism, we investigated whether food antigens participated in the effects of long-term oral alcohol (AL) administration on serum immunoglobulin levels and polyclonal antibody production in spleen, Peyer's patch and bone marrow of C57BL/6 mice in specific pathogen free (SPF) circumstances. In animals fed antigen food (AF), three weeks of oral AL administration elicited an increase in polyclonal IgA antibody production in Peyer's patch. Moreover, seven weeks of oral AL administration elicited increases in both polyclonal IgA antibody production and serum IgA level. However, in animals fed antigen free food (AFF), oral AL administration failed to elicite increases in serum IgA level and polyclonal antibody production in spleen, Peyer's patch and bone marrow. Furthermore, in animals fed AF or AFF, oral AL administration failed to elicite increases in polyclonal IgM and IgG antibody production in any organ and serum IgM and IgG level. It is suggested that food antigens participated greatly in the elevation of serum IgA level in chronic alcoholism and that Peyer's patch is the major site of polyclonal IgA antibody production.     

Experimental IgA nephropathy induced by oral immunization

To test the hypothesis that IgA nephropathy can result from a mucosal immune response, mice were orally immunized with one of three protein antigens for 14 wk. Such mice exhibited an essentially pure mucosal antibody response characterized by specific IgA-producing plasma cells in exocrine sites and specific IgA antibodies in serum. Furthermore, 73% of immunized mice had IgA and 88% had immunogen deposited in the glomerular mesangium, and 64% of immunized mice examined ultrastructurally had electron-dense mesangial deposits. All three were present concurrently in 57% of the immunized mice. No differences in regard to IgG or IgM were observed between immunized and control mice for any of these parameters. Mucosal immunization therefore can result in a specific immune response that leads to mesangial deposition of immune complexes containing IgA antibody. In its fundamental features the experimental renal lesion resembles that seen in the human disease IgA nephropathy.     

Clearance of circulating IgA immune complexes is mediated by a specific receptor on Kupffer cells in mice

To characterize the physiology of circulating IgA immune complexes (IgA- IC), the dynamics of IgA-IC removal by the liver were examined. After intravenous injection, covalently cross-linked IgA antibodies to the dinitrophenyl determinant were rapidly removed from the circulation by the liver. Immunofluorescence microscopy and light and electron microscope autoradiography showed that the IgA-IC were associated with Kupffer cells. With increasing doses of injected IgA-IC the clearance velocity approached a maximum, thus prolonging the circulation of IgA- IC. All these observations indicated a receptor-mediated process. Saturating doses of various potential receptor-blocking agents, heat- aggregated mouse IgG, microaggregated human serum albumin, and purified dimeric IgA did not influence the clearance pattern and hepatic uptake of radiolabeled IgA-IC. Mouse livers were also perfused via the portal vein with 1 microgram of IgA-IC. In the presence or absence of serum proteins, 43% of the perfused IgA-IC were removed in a single passage. This liver uptake was not reduced with simultaneous perfusion of large doses of aggregated mouse IgG, aggregated human serum albumin, or purified free dimeric mouse IgA. In contrast, the liver uptake of radiolabeled IgA-IC was decreased by 88% with the addition of 1 mg unlabeled IgA-IC. These observations support the conclusion that removal of IgA-IC from circulation is mediated by a specific IgA receptor on Kupffer cells.     

Class- and subclass-specific antibody response to hemocyanin in nonmalignant paraproteinemia.

IgM, IgG, and IgA class-specific, as well as IgG subclass-specific antibody titers against the primary immunogen HPH were measured with ELISA in 19 patients with nonmalignant paraproteinemia (eight with IgG1, two with IgG2, two with IgG4, four with IgM, and three with IgA) and in a simultaneously studied age- and sex-matched control group. After primary immunization only IgM and IgA anti-HPH titers were significantly lower in the patient group. Four patients with relatively high IgG or IgA serum paraprotein levels did not produce antibodies in some Ig classes or IgG subclasses, whereas all other patients and all controls developed antibody titers in all classes and IgG subclasses. Low or absent antibody titers did not occur preferentially in the Ig (sub)classes to which the paraproteins belonged. After secondary immunization the patients could not increase or maintain their antibody titers as well as the controls, and this was most clear in the IgM and IgA antibody class. A direct correlation between polyclonal serum IgM levels and IgM anti-HPH titers was present in the patients. Such a correlation was absent for IgA in the patients and for all classes in the controls. It is concluded that humoral immunosuppression as measured with a newly encountered antigen in patients with nonmalignant paraproteinemia is most clearly expressed in the IgM and IgA antibody class and that the paraprotein (sub)class is not preferentially involved.     

Immunoblotting is of no value in the serodiagnosis of Streptococcus pneumoniae pneumonia

We studied five patients with bacteremic pneumococcal pneumonia to determine if Western blot analysis of the immune response to protein antigens of this microorganism could be used as a diagnostic tool on serum samples from patients with pneumonia of unknown etiology. The immune response to capsular polysaccharide (CP) was determined by Dr. G. Schiffman. All five patients had an increase in antibody to the infecting capsular polysaccharide type between the acute and convalescent serum samples. Depending on the antibody (polyvalent, IgM, IgA, IgG) and the sample tested (acute or convalescent), a number of protein antigens were recognized: polyvalent conjugate, 14–25 antigens with acute phase serum samples and 18–27 antigens with convalescent serum samples; IgM, 8–9 antigens with acute phase serum samples and 10–14 with convalescent phase serum samples; IgA, 2–11 antigens with acute phase serum samples and 10–12 protein antigen with convalescent phase samples; IgG, 6–14 antigen with acute phase and 17–19 antigens with convalescent phase serum samples. Antibodies were directed towards proteins with molecular weights ranging from 14,300 – 200,000 Da. There were no unique antibodies detected in the convalescent serum samples. We conclude that Western immunoblotting is of no value as a diagnostic tool for pneumococcal pneumonia.     

Immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen

The immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen is described. Serum samples containing antibody IgM to HIV-1 p17 antigen were incubated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 (rp17) conjugate and rp17-β-D -galactosidase conjugate, and the immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. Subsequently, the immune complex was transferred to white polystyrene beads coated with monoclonal mouse (antihuman IgM) IgG in the presence of excess of ϵN-2,4-dinitrophenyl-L -lysine. The signal for antibody IgM to p17 antigen was the fluorescence intensity by fluorometric assay of β-D -galactosidase activity bound to the white polystyrene beads. The periods of time required for the formation, trapping, and transferring of the immune complex comprising the three components were more than 4 hr, 2 hr, and 3 hr, respectively. The immunoassay developed was shown to be specific by inhibition of transferring the immune complex in the presence of excess of nonspecific IgM but not IgG. Signals for antibody IgM to p17 antigen with serum samples of HIV-1 seroconversion serum panels,—that is, with serum samples in early stages of the infection—tended to be higher than those with serum samples from HIV-1 asymptomatic carriers probably long after the infection and patients with ARC and AIDS. In contrast, signals for antibody IgG to p17 antigen with serum samples of HIV-1 seroconversion serum panels tended to be higher than signals for antibody IgM to p17 antigen but were much lower than signals for antibody IgG to p17 antigen with serum samples from HIV-1 asymptomatic carriers and patients with ARC and AIDS. J. Clin. Lab. Anal. 12:329–336, 1998. © 1998 Wiley-Liss, Inc.     

Deficiency in serum immunoglobulin (Ig)M predisposes to development of IgG autoantibodies

Serum immunoglobulin (Ig)M provides the initial response to foreign antigen and plays a regulatory role in subsequent immune response development, accelerating the production of high-affinity IgG. Here we show that mice deficient in serum IgM have an increased propensity to spontaneous autoimmunity as judged by the development with age of serum IgG anti-DNA antibodies and the renal deposition of IgG and complement. They also exhibit augmented anti-DNA IgG production on exposure to lipopolysaccharide. Thus, deficiency in serum IgM leads to diminished responsiveness to foreign antigens but increased responsiveness to self-a paradoxical association reminiscent of that described in humans deficient in complement or IgA.We wondered whether serum IgM might play an analogous role with regard to the response to self-antigens. However, here-in contrast to the sluggish response to foreign antigens-we find that deficiency in serum IgM actually predisposes to the development of IgG antibodies to autoantigens.     

Rapid active transport of immunoglobulin A from blood to bile

Immunoglobulins were isolated from the serum or ascitic fluid of Lou/Wsl rats bearing plasmacytomas and labeled with 125I. When labeled IgA was injected i.v. it disappeared from the blood serum much more rapidly than IgG2 so that after 3 h less than 10% remained. This rapid disappearance of the injected IgA was not seen in rats with ligated bile ducts. In rats with cannulated bile ducts, the labeled IgA appeared rapidly in the bile so that 25% of the injected dose was recovered in 3 h; at the peak of this biliary excretion the specific radioactivity of the bile (cpm/milligram protein) was about 200 times greater than that of the blood serum. Thus much of the IgA which finds its way into the blood is rapidly and actively transported across the liver so that it enters the gut lumen via the biliary tract.     

Rheumatoid factor (RF) production during anamnestic immune responses in the mouse. III. Activation of RF precursor cells is induced by their interaction with immune complexes and carrier-specific helper T cells

IgG1 immune complexes were identified as the humoral stimuli responsible for the synthesis of IgG1-specific IgM rheumatoid factor (RF), which occurs in the mouse during the early stages of secondary immune responses to protein antigens. The specificity of this phenomenon was illustrated by the fact that complexes made with IgG1 F(ab')2 fragments or with antibodies of a different isotype failed to induce significant anti-IgG1 RF synthesis. The importance of immune complexes in the induction of RF was further underscored by the substantial increase in the titers of isotype-specific RF observed in the serum of mice immunized with IgG1- or IgG2a-complexed antigen rather than with antigen alone. The RF-inducing capacity of the complexes varied with the antigen/antibody ratio: it was maximal in antibody excess or at equivalence, but dramatically reduced in large antigen excess. The importance of T cell priming in RF precursor cell activation by immune complexes was demonstrated by the failure of T cell-deprived spleen cells to reconstitute the capability of irradiated mice to produce RF, and by the optimal RF responses observed after reconstitution of irradiated recipients with primed T cells and naive B cells. The involvement of T cells in this process could not be explained by the release of nonspecific B cell activators, because antigenic stimulation of primed T cells failed to enhance the activation of RF precursor cells by immune complexes of unrelated antigen.     

Enzymolysis of glomerular immune deposits in vivo with dextranase/protease ameliorates proteinuria, hematuria, and mesangial proliferation in murine experimental IgA nephropathy.

The therapeutic effects of saccharolytic and proteolytic enzymes were investigated in models of IgA nephropathy. Mesangial glomerulonephritis was induced in mice by intravenous injection of preformed soluble immune complexes of dextran sulfate and either IgA (J 558) or IgM (MOPC 104 E) anti-dextran MAb (passive model) or by immunization with DEAE dextran (active model). In the passive model, only 30-40% of dextranase-treated mice given IgA or IgM immune complexes had mesangial Ig or dextran deposits, compared with 100% of saline-treated controls (P less than 0.01). There was no significant difference in mice given only protease. In the active model, dextranase and protease separately each reduced glomerular dextran and C3 deposits, and hematuria (P less than 0.01). Dextranase also reduced the glomerular IgA deposits (20 vs. 100% of saline-treated mice) and the frequency and severity of mesangial matrix expansion (both P less than 0.02), but did not reduce the modest IgG or IgM codeposits. Protease reduced IgG and IgM deposits, proteinuria and mesangial hypercellularity compared with saline (P less than 0.02), but did not diminish IgA, and had no effect on mesangial matrix expansion. The combination of dextranase plus protease attenuated all components of glomerular injury as judged by clinical and pathological parameters, but inactivated dextranase plus inactivated protease had no effect on any parameter. We conclude that enzymatic digestion of antigen and antibody can reduce immune deposits, mesangial proliferation, proteinuria, and hematuria in experimental glomerulonephritis.     

Natural antibodies in sera of patients with systemic lupus erythematosus

Sera obtained from fifty-five patients with active systemic lupus erythematosus (SLE) and from four patients with mixed connective tissue disease (MCTD) previously shown by immunofluorescence and by double immunodiffusion to possess antinuclear antibodies, were tested for the presence of natural antibodies of IgG, IgA, and IgM isotypes. Antibody activity to actin, myosin, DNA, TNP, albumin, and tubulin was examined, using an enzyme-linked immunosorbent assay (ELISA). It was found that, in comparison with the antibody titers in normal sera, most of the SLE and MCTD sera possessed statistically greater amounts of IgG, IgA, and IgM antibodies directed against all the antigens tested. Furthermore, the IgG, IgA, and IgM antibody activity to DNA and TNP, compared to that found with all the other antigens, was significantly higher. Antibodies reacting with a saline extract of calf thymus (ECT) were studied by ELISA and by immunodiffusion. No correlation was observed between the natural antibody titers and the serum antibody levels to ECT detected either by ELISA or by immunodiffusion.     

Serodiagnosis of herpes encephalitis by indirect enzyme-linked immunosorbent assay,experience from a Swedish antiviral trial

Analysis of IgM/IgG/IgA antibody class activity to herpes simplex (HSV), measles and varicella zoster (VZV) antigen by indirect enzyme-linked immunosorbent assays has been applied to the cerebrospinal fluid and serum from 127 cases of focal encephalitis—39 patients with and 88 patietns without biopsy/necropsy.Results indicative of herpes etiology were found in 53 cases. Intrathecal antibody production was found in all CSF samples taken beyound the tenth day after the onset of neurological illness with one exception—a boy treated with acyclovir for a primary infection within 20 hours after the onset of illness. Intrathecal HSV antibody response was of IgG class in 94%, IgM class in 70% and IgA in 94%.In primary infections, the HSV IgM CSF response was high and contributed to an early diagnosis; in recurrent infections it was often at a low level and late. HSV IgM persisted from 60 to 328 days, IgG and IgA during observation times to 678 days. Cross-reactivity with varicellae zoster was found to be restricted to IgG only.     

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice

The IgM antibody response of mice to the streptococcal group A carbohydrate (GAC) was measured. With most strains tested, large amounts of IgM antibody were produced; in AKR mice, over 1% of the total nucleated spleen cells secreted IgM anti-GAC antibody after hyperimmunization. The relative avidity of the antibody was extimated by a modification of the Jerne plaque assay where spleen cells from individual mice were tested against erythrocytes with varying GAC epitope densitymthese studies showed that the earliest, as well as latest, IgM antibodies produced were highly restricted in avidity heterogeneity. No evidence of affinity maturation was seen upon hyperimmunization. These data favor the conclusion that the restricted IgG response seen in mice hyperimmunized to GAC is not the result of affinity driven competition for antigen among precursor cells.     

gamma/delta T cell-deficient mice have impaired mucosal immunoglobulin A responses

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCRdelta-/-) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCRdelta-/- mice. The TCRdelta-/- mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toxoid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toxoid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody- producing cells were present in the intestinal lamina propria and Peyer's patches of TCRdelta-/- mice compared with the orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta cells in mucosal immunity.     

Antibiodies of the IgG, IgM, and IgA classes in newborn and adult sera reactive with gram-negative bacteria

Umbilical cord serum and adult serum antibodies reactive with heat-stable somatic antigens of Gram-negative bacteria (Neisseria gonorrhoeae, Escherichia coli, and Salmonella typhosa) were assayed by using an indirect fluorescent antibody test. Reactive IgG, IgM, and IgA antibodies were identified by using fluoresceinconjugated antisera specific for these immunoglobulin classes.IgG antibody titers in cord serum approximated those found in the corresponding maternal sera. IgM and IgA antibodies were present in adult sera but were not demonstrable or were present only in small amounts in cord sera. The presence of IgG and IgM antibodies reactive with Gram-negative bacteria was confirmed by the testing of purified 7S and 19S fractions. In addition, both IgG and IgM reactivities were inhibited by the prior incubation of serum with purified specific lipopolysaccharide preparations.The ubiquity and magnitude of these natural IgG antibodies in the sera of both adults and neonates have apparently eluded detection in previous studies. The use of bactericidal and agglutination tests, which are apparently more sensitive to the presence of IgM than to IgG antibodies, may account for the failure of previous studies to detect adult and cord IgG antibodies reactive with somatic antigens of Gram-negative bacteria. The presence of these IgG antibodies may be correlated with the resistance to infection demonstrated by most newborns as they are challenged by the septic extrauterine environment.     

Secretory component as the receptor for polymeric IgA on rat hepatocytes

Rat hepatocytes in short-term monolayer cultures bound radiolabeled polymeric rat IgA but not IgG. The binding of 125I-IgA was inhibited equally well by unlabeled polymeric IgA and by antiserum to rat secretory component (SC). The antibody to SC, after specific purification and radiolabeling, was bound to hepatocytes as effectively as the IgA. These results indicate that SC acts as the receptor for polymeric IgA on rat hepatocytes as it does on human gut epithelia, and that the transport of IgA from blood to bile in rats across the liver is analogous to that of IgA across human enterocytes.     

Altered hepatic transport of immunoglobulin A in mice lacking the J chain

We have created J chain knockout mice to define the physiologic role of the J chain in immunoglobulin synthesis and transport. The J chain is covalently associated with pentameric immunoglobulin (Ig) M and dimeric IgA and is also expressed in most IgG-secreting cells. J chain- deficient mice have normal serum IgM and IgG levels but markedly elevated serum IgA. Although polymeric IgA was present in the mutant mice, a larger proportion of their serum IgA was monomeric than was found in wild-type mouse serum. Bile and fecal IgA levels were decreased in J chain-deficient mice compared with wild-type mice, suggesting inefficient transport of J chain-deficient IgA by hepatic polymeric immunoglobulin receptors (pIgR). The pIgR-mediated transport of serum-derived IgA from wild-type and mutant mice was assessed in Madin-Darby canine kidney (MDCK) cells transfected with the pIgR. These studies revealed selective transport by pIgR-expressing MDCK cells of wild-type IgA but not J chain-deficient IgA. We conclude that although the J chain is not required for IgA dimerization, it does affect the efficiency of polymerization or have a role in maintaining IgA dimer stability. Furthermore, the J chain is essential for efficient hepatic pIgR transport of IgA.     

Immune response to bacterial dextrans. II. T cell control of antibody isotypes

The isotype distribution of Dextran B 512 (Dex)-specific plaque-forming cells (PFC) and serum antibodies was studied after in vivo immunization in C57BL/6 mice. Although IgG2b and IgG3 could also be detected in most individuals, the majority of non-IgM PFC were of the IgA isotype. All classes other than IgM were T cell-dependent, as shown by their complete absence in athymic "nude" mice. This unusual isotype pattern was further investigated by studying the antibody responses to the same Dex epitope coupled to a protein carrier, and to a different hapten coupled to the carrier Dex or to a protein. The results show that IgA responses are epitope-related and selectively associated with anti-Dex antibodies: no IgA PFC are detected against a hapten coupled to Dex or proteins, while the enhanced levels of helper cell reactivity provided by protein carrier to Dex result in the appearance of IgG1 antibodies in addition to IgA. These results indicate that T cells that modulate isotype patterns in these responses can discriminate between Dex- and DNP-specific B cells in the response to the same carrier. Since the same idiotype is detected on a large fraction of the IgM and IgA anti-Dex response and antiidiotypic helper cells have previously been detected in normal C57BL/6 mice, we suggest that idiotype-specific T cells control the production of IgA antibodies upon immunization with Dex.     

Systemic immune response to oral polio immunization in patients with IgA nephropathy

The systemic immune response to a booster dose of attenuated polio vaccine has been studied in a group of patients with IgA nephropathy, their parents and healthy unrelated controls. The serum IgA and IgG antibody response and the antibody response in cultured lymphocyte supernatants was quantitated by a sensitive radio-immunoassay. Three of eight patients and two of their parents had elevated serum IgA antibody to polio pre-immunization. There was no significant rise in serum IgA or IgG antibody to polio post-immunization in any group. However, the five subjects with elevated pre-immunization levels fell into the normal range post-immunization, suggesting IgA specific suppression. Total and antigen specific IgA rose post-immunization in culture supernatants in all groups. The rise did not differ among the groups. The patients and parents with high serum antibody also had high culture supernatant levels and these fell post-immunization. There was an increased antigen non-specific IgG response in four patients and three of their parents. The data provide additional in vivo evidence of an upregulated IgA immune response in patients with IgA nephropathy. In addition, the data provide the first in vivo evidence of aberrant immune responses in the first degree relatives of patients with IgA nephropathy. This appears to be one of perhaps several inherited traits which may predispose an individual to develop this common and progressive nephropathy.