Increased serum albumin,gamma globulin,immunoglobulin IgG,and IgG2 and IgG4 in autism

BACKGROUND: Research on the biological pathophysiology of autism has found some evidence that immune alterations may play a role in the pathophysiology of that illness. As a consequence we expected to find that autism is accompanied by abnormalities in the pattern obtained in serum protein electrophoresis and in the serum immunoglobulin (Ig) and IgG subclass profile. METHOD: We examined whether subjects with autism showed changes in total serum protein (TSP) and the serum concentrations of albumin, alpha1 globulin, alpha2 globulin, beta globulin and gamma globulins, IgA, IgM and IgG and the IgG subclasses IgG 1, IgG2, IgG3 and IgG4, compared with normal controls. RESULTS: We found significantly increased concentrations of TSP in autistic subjects, which were attributable to increased serum concentrations of albumin and gamma globulin. Serum IgG, IgG2 and IgG4 were also significantly raised. In autism there were significant and positive correlations between social problems and TSP and serum gamma globulin and between withdrawal symptoms and TSP and serum albumin and IgG. CONCLUSIONS: The results suggest that autism is characterized by increased TSP, a unique pattern obtained in serum protein electrophoresis, i.e. increased serum albumin and IgG, and by a specific IgG subclass profile, i.e. increased serum IgG2 and IgG4. The increased serum concentrations of IgGs in autism may point towards an underlying autoimmune disorder and/or an enhanced susceptibility to infections resulting in chronic viral infections, whereas the IgG subclass skewing may reflect different cytokine-dependent influences on autoimmune B cells and their products.     

Fc gamma receptor recognition of IgG ligand by human monocytes and macrophages

We examined the binding characteristics of human monocytes and macrophages with the IgG ligands, human monomeric IgG and a small human IgG aggregate, trimeric IgG. Our purpose was to utilize fresh monocytes, in vitro cultured monocytes, and alveolar macrophages in direct and indirect binding experiments. Freshly isolated monocytes expressed only a single binding site for IgG monomer and IgG trimer. In contrast, in vitro cultured monocytes, gamma-interferon-treated monocytes, and freshly isolated alveolar macrophages expressed a single binding site for IgG monomer and, in addition, a high and low affinity binding site for IgG trimer. The high affinity binding site for IgG trimer (Kd approximately equal to 1 nM) appeared identical to the binding site for IgG monomer. The low affinity binding site for IgG trimer (Kd = 50 to 250 nM) appeared to be due to Fc gamma RII, because antibody to Fc gamma RII inhibited its expression. Since Fc gamma RII, in contrast to Fc gamma RI, does not bind monomeric IgG, the data suggest that this low affinity receptor for trimeric IgG, Fc gamma RII, can bind low molecular weight circulating immune complexes at concentrations 10- to 100-fold lower than Fc gamma RI. Thus, these studies suggest that at 37 degrees C, macrophage Fc gamma RII may play a functional role in the recognition of small molecular weight immune complexes.     

Signal transduction via both human low-affinity IgG Fc receptors, Fc gamma RIIa and Fc gamma RIIIb, depends on the activity of different families of intracellular kinases

In contrast to IgG Fc receptor II (Fc gamma RIIa), the function of Src-family kinases, Syk and phosphoinositide 3-kinase (PI3K) in signal transduction of glycosylphosphatidylinositol-anchored Fc gamma RIIIb has not been analyzed in detail. Therefore pharmacological inhibitors were used to define the role of specific kinases in signalling of Fc gamma RIIa and Fc gamma RIIIb in myeloid cells. We demonstrate that the broad tyrosine kinase inhibitor genistein, the Src-family kinase inhibitor PP2, and the Syk kinase inhibitor piceatannol inhibit [Ca2+]i rise induced by both low-affinity Fc gamma R in neutrophils. Genistein and PP2 additionally prevent Fc gamma R-triggered hydrogen peroxide generation. In contrast, wortmannin, a PI3K inhibitor, which blocks Fc gamma RIIIb activation completely, abolishes Fc gamma RIIa-mediated [Ca2+]i flux only in the beginning. In addition, herbimycin A, a further specific inhibitor of Src-family kinases leads to a delayed Fc gamma RIIa-induced [Ca2+]i rise in THP-1 cells. In summary, our data demonstrate differences between both low-affinity IgG Fc receptors, and provide evidence for an essential role of Src-family kinases, Syk and PI3K in Fc gamma RIIIb-mediated signalling.     

Antitrichomonas IgG,IgM, IgA,and IgG subclass responses in human intravaginal trichomoniasis

Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is a major nonviral sexually transmitted disease. Clinical spectrum varies from an asymptomatic state to mild, moderate, or severe symptoms. However, the exact factors leading to the variations in symptoms have not been well elucidated. Host’s immune response to the parasite may be playing a role in varied symptomatology. The present study reports antitrichomonas IgM, IgA, IgG and its subclasses in doubling dilutions of serum and diluted vaginal washes of six T. vaginalis-infected symptomatic and four T. vaginalis-infected asymptomatic women and uninfected controls by enzyme-linked immunosorbent assay (ELISA). No significant difference was observed in serum IgG ELISA absorbance values from symptomatic compared to asymptomatic subjects (p > 0.05) while a significant difference (p < 0.05) was noted in serum IgM in all the tested dilutions and IgA up to a dilution of 400. This is the first report of the detection of specific IgG subclass response in T. vaginalis-infected female patients, and quantitative analysis of the antibody responses indicated that the production of local IgG particularly IgG1 in vaginal secretions may be playing a significant role in establishing symptomatic infection. The interesting observation of the present study is that the specific IgM was detected in 2 (33.3%) symptomatic and T. vaginalis-infected patients in ≥800 dilutions and in 1 (16.6%) up to 200 dilutions in serum, while it was not detectable in the vaginal secretions of symptomatic patients or in the serum and vaginal secretions of asymptomatic T. vaginalis-infected patients.     

Quantitation of human total IgG, kappa IgG and lambda IgG in serum using monoclonal antibodies

Monoclonal antibodies to human IgG have been applied, in parallel with polyclonal antisera, to the routine quantitation of human IgG. Two monoclonal antibodies directed against spatially distinct epitopes have been used in combination to form insoluble complexes exhibiting turbidity. Quantitation was performed using a centrifugal fast analyser and a correlation coefficient of 0.979 was obtained for the two estimations. The technique has been further developed to allow the separate quantitation of kappa IgG and lambda IgG in whole serum.     

Distinctive role of IgG1 and IgG3 isotypes in Fc gamma R-mediated functions

Polyclonal and monoclonal anti-Rh (D) antibodies of IgG1 and IgG3 subclass were evaluated for their capacity to sensitize erythrocytes and (i) to trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC); (ii) to mediate binding to monocyte and lymphocyte Fc gamma R; (iii) to stimulate phagocytosis by monocytes. All antibodies were equally effective in mediating monocyte or activated U937 cell ADCC but IgG1 was more active than IgG3 in K-cell mediated ADCC. IgG3-sensitized erythrocytes inhibited IgG1-induced lysis, suggesting that each subclass engages the same Fc gamma R receptor but that lysis requires a further 'signal' that the IgG3 molecule can not deliver. Two monoclonal IgG3 anti-D antibodies were shown to have higher binding (two times) and phagocytic (three times) indices than IgG1 antibody for monocytes; similar differences were observed for polyclonal IgG1 and IgG3 antibodies. The same pattern was observed in an EA rosette assay when a total lymphocyte population was used; however, this difference was not seen with a B-cell depleted (T+ null cell) lymphocyte population.     

Functional mapping of the Fc gamma RII binding site on human IgG1 by synthetic peptides

Receptors specific for the Fc part of IgG (Fc gamma R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc gamma R may result in autoimmunity. Thus, the modulation of IgG-Fc gamma R interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to Fc gamma RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P(234) and S(298) were synthesized and used in binding and functional experiments. Binding of the peptides to Fc gamma R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T(256)-P(271), which bound to soluble recombinant Fc gamma RIIb with K(d)=6 x 10(6) M(-1). The biotinylated peptides R(255)-P(271) and T(256)-P(271) complexed by avidin exhibited functional activity; they induced Fc gamma RIIb-mediated inhibition of the BCR-triggered Ca(2+) response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF-alpha and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc gamma RII-binding part of IgG1.     

Bovine IgG can aggregate at conditions simulating pasteurization and binds to some human Fc gamma receptors

The ability of bovine IgG preparations to bind to the various distinct human leukocyte Fc gamma receptors was studied. In experiments using intact cells and isolated Fc gamma receptors, it was demonstrated that bovine IgG can bind to Fc gamma receptors of four human cell types but not to Fc gamma receptors of human neutrophils. 125I-labeled Fc gamma receptors purified on human IgG-Sepharose columns from human B and T lymphocytes, monocytes and eosinophils were able to rebind specifically to insolubilized bovine IgG. In contrast, radioiodinated human neutrophil Fc gamma receptors did not rebind to bovine IgG-Sepharose. A similar pattern of specificity was demonstrated in studies of the binding of 125I-labeled heat-aggregated bovine IgG to various human leukocyte populations. The labeled aggregated bovine IgG bound to peripheral blood mononuclear cells, to B cells from chronic lymphocytic leukemia patients and to macrophage-like U-937 cells, but bound poorly to normal human granulocytes. Labeled non-aggregated bovine IgG was not appreciably bound to any of the cell populations. Since bovine IgG in dietary sources is frequently exposed to heat, the effect of heating on the physical state and Fc-binding properties of bovine IgG was examined. The data show that heating bovine IgG at concns of 0.9-3.6 mg/ml at 63 degrees C for 30 min in neutral buffer causes aggregation of bovine immunoglobulin (10-16% aggregation) and increases the ability of bovine IgG preparations to bind to human Fc gamma receptors of intact cells. Gel filtration studies suggest the possibility that bovine IgG may also be aggregated during the pasteurization of raw milk.     

Analysis of Ig subclass deficiency: First reported case of IgG2, IgG4, and IgA deficiency caused by deletion of C alpha 1, psi C gamma, C gamma 2, C gamma 4, and C epsilon in a Mongoloid patient

BACKGROUND: IL-4 and IL-13 have been shown to be critical for expression of the asthma phenotype in a murine model and may modulate human fibroblast function. OBJECTIVE: We hypothesized that IL-4 and IL-13 would increase airway fibroblast proliferation and reduce the ability of dexamethasone to decrease this proliferation. METHODS: Six subjects with severe asthma, 5 subjects with mild asthma, and 5 healthy subjects underwent bronchoscopy with endobronchial biopsy. Biopsy specimens were placed in Dulbecco modified Eagle medium and cultured, and only fibro-blasts from the first and second passages were evaluated. Cells were incubated with IL-4 (50 ng/mL), IL-13 (10 ng/mL), and the combination for 48 hours in the presence and absence of dexamethasone, 10(-7) mol/L, and 10(-8) mol/L. Fibroblasts were also incubated with IFN-gamma at 50 ng/mL to assess the response of a T(H)1 cytokine on proliferation. RESULTS: Fibroblast proliferation, determined by (3)H-thymidine incorporation, was significantly increased by IL-4 in subjects with mild asthma as compared with IL-4 in subjects with severe asthma and healthy subjects (P =.003), IL-13 (P =.011), and the combination (P =.004). Dexamethasone also increased proliferation in the group with mild asthma as compared with the group with severe asthma and the healthy group (10(-7) mol/L, P =.02; 10(-8) mol/L, P =.02). IFN-gamma did not significantly alter airway fibroblast proliferation. CONCLUSION: IL-4, IL-13, and dexamethasone all significantly increased fibroblast proliferation in subjects with mild asthma.     

Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes.

The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37 degrees to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1.5 x 10(3) anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it.     

The binding affinities of rheumatoid factors interacting with the C gamma 3 homology region of human IgG

The binding affinities of rheumatoid factors interacting with Gm(a), Gm(x), `non a'' and `γ4 non a'' antigens have been measured by an equilibrium molecular sieving technique using rheumatoid sera of known specificity and pFc'' fragments (≡Cγ3 homology region) from IgG molecules of defined subclass and allotype. All the rheumatoid factors studied showed specific binding with the fragments possessing the homologous antigen. The binding affinities of these rheumatoid factors were low, between 10⁴ and 10⁵ litres/mole. In contrast, no binding occurred between rheumatoid factors and fragments lacking the appropriate antigenic determinant.     

Molecular interactions between human IgG, IgM rheumatoid factor and streptococcal IgG Fc receptors

Group A streptococci type M15 were previously shown to bind both human IgG via the Fc component and a purified monoclonal IgM kappa rheumatoid factor (IgM RF). Using 125I-labelled IgG and 125I-labelled IgM RF, the present study gave association constants of 2.2 x 10(7) and 2.9 x 10(8) M-1, respectively. The binding of 125I-IgG to the streptococci was inhibited by unlabelled IgG, IgG Fc and fragment D of staphylococcal protein A but not by the IgM RF or F(ab')2 of anti-idiotype antibodies to RF (anti-Id RF). Inversely, unlabelled IgM RF and anti-Id RF inhibited the binding of 125I-IgM RF markedly and unlabelled human IgG and IgG Fc only slightly or moderately, respectively. Thus, group A streptococci type M15 showed different binding sites for IgG Fc and the antibody combining sites of a human monoclonal RF. The findings were still more complex on a background of previous reports showing that streptococcal IgG Fc receptors and RFs bind to the same amino acids on the Fc molecule. This complex pattern may play a role in the pathogenesis of rheumatoid arthritis.     

The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding.

We have found that amino acid residues necessary for C1q and Fc gamma R binding of human IgG1 are located in the N-terminal region of the CH2 domain, residues 231-238, using a matched set of engineered antibodies based on the anti-HLA-DR antibody L243. Changing the leucine 235 in the CH2 region of IgG3 and IgG4 to glutamic acid was already known to abolish Fc gamma RI binding. We have confirmed this for IgG1 and also found a concomitant abolition of human complement lysis with retention of Fc gamma RIII-mediated function. Changing the glycine at 237 to alanine of IgG1 also abolished Fc gamma RI binding and reduced human complement lysis and Fc gamma RIII-mediated function. Exchanging the whole region 233-236 with the sequence found in human IgG2, abolished Fc gamma RI binding and human complement lysis and reduced Fc gamma RIII-mediated function of IgG1. In contrast, a change in the previously described C1q-binding motif, from lysine at 320 to alanine, had no effect on IgG1-mediated complement lysis.     

Simultaneous absence of the human IgG1, IgG2, IgG4 and IgA1 subclasses: Immunological and immunogenetical considerations

Simultaneous absence of the IgG1, IgG2, IgG4 and IgA1 immunoglobulins has been unambiguously demonstrated in a healthy 75-year-old woman by testing for allotypes, isoallotypes and for isotypes of these four subclasses. Only IgM, IgD, IgG3, IgA2 and IgE were present. The IgG3 levels were significantly increased. Family investigation showed inheritance of a haplotype Gm^?;?;b A2m². This person is homozygous for an extensive DNA deletion including the Cγ1, Cγ2, Cγ4 and Cα1 genes.     

Role of neutrophil Fc gamma RIIa (CD32) and Fc gamma RIIIb (CD16) polymorphic forms in phagocytosis of human IgG1- and IgG3-opsonized bacteria and erythrocytes.

The four subclasses of IgG have different biological activities associated with their Fc regions. Fc gamma receptors on leucocytes (Fc gamma R) mediate binding and phagocytosis of opsonized particles. Two structurally and functionally distinct allelic polymorphisms of the Fc gamma R have been defined: the H/R131 forms of Fc gamma RIIa (CD32), and the neutrophil antigen 1 (NA1)/NA2 forms of Fc gamma RIIIb (CD16). In this study the activities of allotypes of CD16 are analysed with antibacterial IgG subclass antibodies and with IgG1 and IgG3 anti-Rhesus D, and the activities of CD32 with IgG1 and IgG3 anti-Rhesus D. With respect to the allotypes of CD16, polymorphonuclear leucocytes (PMN) homozygous for Fc gamma RIIb-NA2 exhibited a lower (21-25%) IgG1-mediated phagocytosis of Staphylococcus aureus strain Wood (STAW), Haemophilus influenzae type b (Hib), and Neisseria meningitidis group B (NMen) than IIIb-NA1 PMN. The difference was apparent only when the micro-organisms were opsonized in the absence of complement, and was furthermore enhanced (34-52%) upon blockade of Fc gamma RIIa. In addition, monoclonal IgG3 anti-D-mediated rosette formation and phagocytosis was consistently found to be lower (16%) with Fc gamma RIIIb-NA2 than with IIIb-NA1 PMN. For the allotypes of CD32 we now show that IgG3 anti-D sensitized erythrocytes formed more (50%) rosettes and were phagocytosed at a higher rate with PMN carrying Fc gamma RIIa-H131 than with PMN carrying IIa-R131. Heterozygous Fc gamma RIIa-H/R131 PMN exhibited intermediate phagocytic activity in this respect. This study illustrates a critical role of Fc gamma R allotypes in functional interactions with biologically relevant IgG subclass antibodies.     

Bovine IgG1, but not IgG2, binds to human B cells and inhibits antibody secretion

We previously observed that milk-derived bovine IgG, but not serum-derived bovine IgG, strongly inhibits antibody secretion by pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC). Bovine milk contains a greater percentage of IgG1 (90%) than does bovine serum (53%). To determine whether bovine IgG subclasses have different functional capabilities, we have examined the effects of bovine IgG1 and IgG2 subclasses upon not only antibody secretion but also mitogenesis by human PBMC. Both bovine IgG subclasses markedly inhibited PWM-stimulated mitogenesis. However, only bovine IgG1, and not IgG2, inhibited antibody secretion during a 14-day in vitro culture period. Also, antibody secretion was inhibited following a 24-hr preincubation of human PBMC with bovine IgG1, but not with IgG2. To determine whether these differences corresponded to specificities of human Fc gamma receptors on subsets of mononuclear cells, fluorescence-activated cell sorter (FACS) analyses were performed. Both bovine IgG subclasses bound to human monocytes. However, only bovine IgG1 bound to human B cells, and bovine IgG1 bound more avidly to human B cells than did human IgG. One model to explain these findings is that inhibition of mitogenesis may be due to the binding of both bovine IgG1 and IgG2 subclasses to monocytes; whereas subclass-specific inhibition of antibody secretion may result from the selective binding of bovine IgG1, but not bovine IgG2, to B cells. The observation that bovine IgG1 has a greater avidity for human B lymphocyte Fc receptors than human IgG may have important implications for future studies of Fc gamma receptors on human leucocytes.     

The Fc receptor for IgG (Fc gamma RII; CD32) on human neonatal B lymphocytes.

B cells express an Fc receptor for IgG (FcgammaRII; CD32) which is involved in feedback inhibition of antibody production. Engagement of FcgammaRII during ligation of the antigen receptor provides an inhibitory signal. FcgammaRII exists as several isoforms, with FcgammaRIIb (which carries an immunoreceptor tyrosine-based inhibition motif; ITIM) being predominant form on adult B cells. The inhibitory role of FcgammaRIIb may be unhelpful to the infant, since primary exposure to infectious agents is likely to be in the presence of maternal IgG. We hypothesized that neonatal B cells would be less susceptible to feedback inhibition by antibody, either through the expression of activation-competent FcgammaRII isoforms (FcgammaRIIa and FcgammaRIIc) or through reduced expression of the inhibitory FcgammaRIIb isoforms. Cord and adult B cells were examined for expression of FcgammaRII isoforms using monoclonal antibodies and RT-PCR. In vitro assays were performed to assess susceptibility of cord and adult cells to FcgammaRII-mediated suppression. Although there is no phenotypic difference in FcgammaRII expression (FcgammaRIIb predominating on both adult and cord B cells), FcgammaRIIb is expressed at lower levels on cord cells. This quantitative difference in FcgammaRIIb expression may explain the reduced susceptibility of cord B cells to antibody-mediated inhibition observed in these experiments.