Untreated IgG Research Articles

Isotype distribution of mucosal IgG-producing cells in patients with various IgG subclass deficiencies

The subclass distribution of IgG-producing immunocytes was examined by immunohistochemistry in nasal and rectal mucosa of infection-prone patients with untreated IgG subclass deficiencies. Biopsy specimens from the two sites were obtained in 18 clinically and serologically well-characterized adult subjects; only a nasal or rectal sample was available from nine similar patients. Chronic lung disease was common in the patient groups with selective serum IgG1 deficiency and combined IgG1 and IgG3 deficiency, whereas the other categories of patients had mainly upper airway and other mild infections. Serum IgG2 or IgG3 deficiency was usually expressed also at the cellular level in rectal mucosa, and the proportion of rectal IgG1 cells was significantly correlated with the IgG1 level (r = 0.90, P less than 0.001). Likewise, there tended to be a decreased expression of the actual subclass at the cellular level in nasal mucosa of patients with serum IgG1 or IgG2 deficiency. Conversely, the median nasal proportion of IgG3 cells was remarkably unaffected by a deficiency of this subclass in serum and rectal mucosa. Interestingly, these patients rather tended to have raised IgG3 and reduced IgG2 cell proportions in their nasal mucosa, although this apparent local IgG3 compensation was nevertheless strongly correlated with the serum IgG3 level (r = 0.87, P less than 0.002). These disparities may reflect different antigenic and mitogenic exposure of the two tissue sites; for example, a persistent protein bombardment of the nasal mucosa that could conceivably override locally a B cell maturation defect. The possible clinical consequences of such variable mucosal expression of IgG subclass deficiencies remain to be studied.

The Fc Fragment Mediates the Uptake of Immunoglobulin G from the Midgut to Hemolymph in the Ixodid Tick <I>Amblyomma americanum</I> (Acari: Ixodidae)

The phenomenon of immunoglobulin uptake from the midgut into the hemolymph has been observed in both argasid and ixodid ticks, but the mechanism of the uptake is not known. Using capillary feeding of adult Amblyomma americanum (L.) female ticks, we investigated the role of immunoglobulin G Fc and Fab fragments in the uptake process. Intact IgG and fragments labeled with 125I were introduced alone or as a mixture with a 10-fold molar excess of unlabeled compounds into ticks, and then the concentration of the compounds after 3 and 6 h of continuous feeding was assessed in the hemolymph. Our results showed that the uptake of Fc fragments was approximately 4 times higher than Fab fragments at 6 h of the feeding and that Fc but not Fab fragments competed with uptake of intact IgG. Glycosylation of the immunoglobulins did not seem to be required for uptake. There was not a statistically significant difference between the uptake of untreated IgG and IgG treated either with periodate or N-glycanase, and untreated Fc fragments or deglycosylated Fc fragments had equal activity in competition experiments with IgG. The uptake into hemolymph of IgG and Fc fragments, but not Fab fragments, was dependent on the pH of the feeding solution and showed a maximum at approximately pH 7.0. In addition, tick midgut cells bound Fc fragments with high affinity, estimated at 21 nM; the interaction with Fab fragments seemed nonspecific. Our results suggest that Fc fragments are the major determinant for the specific uptake of immunoglobulin G into tick hemolymph.

The Fc Fragment Mediates the Uptake of Immunoglobulin G from the Midgut to Hemolymph in the Ixodid Tick Amblyomma americanum (Acari: Ixodidae)

The phenomenon of immunoglobulin uptake from the midgut into the hemolymph has been observed in both argasid and ixodid ticks, but the mechanism of the uptake is not known. Using capillary feeding of adult Amblyomma americanum (L.) female ticks, we investigated the role of immunoglobulin G Fc and Fab fragments in the uptake process. Intact IgG and fragments labeled with 125I were introduced alone or as a mixture with a 10-fold molar excess of unlabeled compounds into ticks, and then the concentration of the compounds after 3 and 6 h of continuous feeding was assessed in the hemolymph. Our results showed that the uptake of Fc fragments was approximately 4 times higher than Fab fragments at 6 h of the feeding and that Fc but not Fab fragments competed with uptake of intact IgG. Glycosylation of the immunoglobulins did not seem to be required for uptake. There was not a statistically significant difference between the uptake of untreated IgG and IgG treated either with periodate or N-glycanase, and untreated Fc fragments or deglycosylated Fc fragments had equal activity in competition experiments with IgG. The uptake into hemolymph of IgG and Fc fragments, but not Fab fragments, was dependent on the pH of the feeding solution and showed a maximum at approximately pH 7.0. In addition, tick midgut cells bound Fc fragments with high affinity, estimated at 21 nM; the interaction with Fab fragments seemed nonspecific. Our results suggest that Fc fragments are the major determinant for the specific uptake of immunoglobulin G into tick hemolymph.

Aggregation of low affinity IgG receptors induces mast cell adherence to fibronectin: requirement for the common FcR gamma-chain.

Mast cells have been reported to increase at sites of immune complex-induced inflammation where these cells appear to potentiate the inflammatory response. The mechanism by which mast cells accumulate at these sites is unknown. One possibility is that aggregation of low affinity IgG receptors could signal mast cells to adhere to components of the connective tissue matrix. To test this hypothesis, we first added aggregated IgG to a mast cell adhesion assay employing fibronectin as a matrix component and observed an increase in cell adhesion. Even a small amount of aggregated IgG (< 60 ng/ml) demonstrated by fast protein liquid chromatography in untreated IgG preparations was sufficient to increase mast cell adhesion by 100%. We next explored the Fc gamma receptors involved. Fc gammaRII/III, which are receptors for oligomeric IgG and were first verified as present on these mast cells by FACS analysis and immunoprecipitation, signaled mast cells to rapidly adhere to fibronectin when aggregated with the anti-receptor Ab2.4G2. The adhesion process mediated by Fc gammaRII/III was not associated with beta-hexosaminidase release. Bone marrow-cultured mast cells from common gamma-chain deficient mice, unlike mast cells cultured from +/+ mice, did not respond to Fc gammaRII/III aggregation. This demonstrated requirement for a gamma-chain implicates oligomeric Fc gammaRIII in the adhesion process. Thus, aggregation of Fc gammaRIII on mast cells leads to mast cell adhesion, demonstrating a previously unknown biological function for this receptor on mast cells and providing a mechanism for mast cell accumulation in immune complex-dependent inflammation.

Demonstration of neutralizing autoantibodies against IL-1 alpha in sera from patients with rheumatoid arthritis.

We have recently reported the presence of IgG which has a potent inhibitory activity against IL-1 alpha in some sera from patients with rheumatoid arthritis. The mechanism of this inhibition by IgG against IL-1 alpha is now elucidated. IgG with IL-1 alpha-inhibitory activity inhibited the binding of 125I-IL-1 alpha to receptors on rheumatoid synovial cells. In addition, preincubation of synovial cells with the inhibitory IgG did not block the binding of 125I-IL-1 alpha to receptors, suggesting a direct interaction between IgG and IL-1 alpha. To examine which region of the IgG, namely Fab or Fc region, has the inhibitory activity, the IgG was digested with papain, and Fab and Fc fragments were purified. Fab fragments, but not Fc fragments, inhibited both IL-1 alpha-induced thymocyte-proliferation and the binding of 125I-IL-1 alpha to receptors. We further demonstrated that the inhibitory IgG which was bound to protein A Sepharose could bind a significant amount of 125I-IL-1 alpha, whereas only a negligible binding of the radiolabeled ligand was detected when IgG without the inhibitory activity was used as control. Moreover, the binding of 125I-IL-1 alpha to IgG with the inhibitory activity was clearly blocked by Fab fragments of IgG having the inhibitory activity. Finally, affinity-purified IgG over an IL-alpha affinity column showed approximately 100-fold more potent inhibitory activity on IL-1 alpha-induced thymocyte proliferation compared with untreated IgG. From these results, we conclude that IgG molecules with IL-1-alpha-inhibitory activity are neutralizing autoantibodies against IL-1 alpha.

Independent binding of native and aggregated IgG in group A streptococci.

Irrespective of IgG Fc-receptor activity, earlier characterized, many group A streptococci were recently found to bind aggregated IgG Fab and/or light chains. In the present study, binding of glutaraldehyde-aggregated, radiolabelled, intact human IgG (a*IgG) to group A streptococci was tested, and strains representing several M-types were found reactive. In particular, high binding was observed among type M12 strains, earlier found devoid of Fc-receptors for monomeric IgG; accordingly, unlabelled, native IgG had little influence on the binding. The sites binding a*IgG were highly sensitive to trypsin and relatively resistant to heat treatment. The binding to M12 was inhibited by human fibrinogen and, to a lesser extent, by heat-aggregated serum albumin. Rabbit antiserum to M12 was more inhibitory than antiserum to a heterologous type of group A streptococci or normal rabbit serum. Our results indicate that streptococcal M-protein binds a*IgG by a multipoint requiring interaction of low specificity and that previously described Fc-receptors binding native IgG are not involved. For comparison, in Cowan I staphylococci and one strain of group G streptococci tested, high binding of a*IgG was also observed; however, this binding was inhibited by native IgG, indicating that protein A and group G streptococcal Fc-receptor, earlier known to bind untreated IgG, also bound a*IgG.

Factors mediating the binding of immune complexes to cryostat sections of psoriatic lesions.

Sheep erythrocytes sensitized with rabbit IgG, or complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP bound to cryostat sections of psoriatic and other skin lesions (lichen planus, discoid lupus erythematosus, sarcoidosis, and mycosis fungoides). Immune complexes with reduced and alkylated IgG (RA-IgG) reacted with preparations of soluble rheumatoid factors (RF) and with sections of RF-coated sheep erythrocytes equally well as did untreated IgG. However, RA-IgG did not react with sections containing FcR-positive cells. These complexes bound either weakly or not at all to skin sections. IgG inhibited the binding of immune complexes to all skin control sections, while RA-IgG only inhibited the binding of complexes to sections of RF-coated cells. Anti-Clq did not inhibit the binding of immune complexes to any section. Periodic acid and formaldehyde abolished FcR activity, whereas neither the activity of soluble RF nor of RF in sections was affected. Only sections of lesional skin from patients with highly active psoriasis and sarcoidosis showed some binding of immune complexes after treatment with periodic acid or formaldehyde. Apparently, the binding of immune complexes in vitro to sections of psoriatic and other skin lesions is mainly mediated by FcR.

Specificity of receptors for IgG on human cytotoxic plaque-forming mononuclear leukocytes. Induction and inhibition of plaque-forming activity.

Intact rabbit IgG antisheep erythrocyte antibodies, and the corresponding F(ab')2 fragments and partially reduced and alkylated IgG were studied for the capacity to induce cytotoxic plaque formation by normal human mononuclear leukocytes in surface monolayers of sheep erythrocytes. The F (ab')2 fragments did not induce plaque formation, whereas partially reduced and alkylated IgG antibodies had a good plaque-inducing capacity compared to untreated IgG anti-SRBC antibodies. The plaque formation was inhibited by human IgG, but not by IgM, IgA, IgD or IgE. Normal and myeloma IgG in aggregated form gave a stronger inhibition than the corresponding proteins. Strong inhibition was observed with IgG1, IgG3, and IgG4, and with IgG2 after aggregation. Both the Fc and pFc' corresponding to the C terminal domain gave a strong inhibition. Thus, the region of the IgG molecule involved in binding to the Fc receptor of the plaque-forming cells appears to be located within the CH3 domain. These observations, therefore, indicate that the plaque-forming cells are of monocytic origin.

Structural requirements in the Fc region of rabbit IgG antibodies necessary to induce cytotoxicity by human lymphocytes.

Rabbit IgG anti-chicken erythrocyte antibodies were compared with the Fab/c or Facb fragments of IgG and with partially reduced and alkylated IgG for the capacity to induce cytotoxicity by normal human lymphocytes. The Fab/c antibody fragment, which lacks one Fab region, was still able to induce cytotoxicity. In contrast, the Facb antibody fragment, which lacks the C-gamma3 domains, was nearly ineffective in activating the effector cells, whereas intact antibody activity was demonstrated by its ability to inhibit the cytotoxicity induced by unsplit IgG. Similarly, partial reduction and alkylation of the IgG antibodies, under conditions affecting the interchain disulphide bonds only, greatly diminished their ability to induce cytotoxicity, although they effectively inhibited the cytotoxicity induced by untreated IgG. On the basis of these results and previous data, we suggest that the reaction of the Fc region of IgG with the effector cell depends on the integrity of the C-gamma2 domain in the native, divalent state or on the interaction between the C-gamma2 and C-gamma3 domains.

Studies of 125I trace labeling of immunoglobulin G by chloramine-T

Detailed studies of the parameters which effect the trace iodination of IgG with 125I, at levels of 1 atom/4000–10,000 moles IgG, using chloramine-T, are described. The following observations were noted: (1) The reactions, (a) the formation of ‘active iodine’ (H 2OI +) and, (b) the incorporation of ‘active iodine’ into protein, that comprise the over-all iodination of proteins have been studied separately and together. (2) With IgG, pH, 7·0 and 2° were optimal and reproducible for both reactions (a) and (b), and for the over-all reaction. Among the parameters which were found to influence the individual and over-all reactions and which were studied to establish optimal conditions were the concentrations of oxidant, iodide, and protein as well as pH, time, temperature, ionic strength, and particular batch of protein. (3) Different proteins and separate lots of the same protein show variable amounts of residues which are oxidized prior to the incorporation of iodine. (4) No differences in the immunochemical precipitation characteristics were detected between untreated IgG and IgG iodinated by the present method. (5) Calculations of the distribution of IgG species, iodinated at a level of 1 atom iodine per 4000–10,000 moles IgG, shows the population of iodinated molecules to be essentially homogeneous and in all likelihood, monosubstituted. (6) Typical conditions for labeling IgG at a level of approximately 1 atom iodine/5000 molecules IgG are described in detail.