A solid-phase radioimmunoassay for the detection of nRNP immune complexes

We developed a solid-phase radioimmunoassay for complement (C)-fixing nuclear ribonucleoprotein (nRNP):anti-nRNP immune complexes (nRNP ICs). The assay was based on the ability of the C-fixing nRNP ICs to bind strongly to immobilized F(ab')2 anti-C3. The extent of binding was quantified by incubating the C-fixing nRNP ICs bound to anti-C3 with 125I-labeled anti-nRNP-specific IgG. The interaction between anti-C3 and C-fixing nRNP ICs was rapid, time- and concentration-dependent and sensitive over a broad range of nRNP IC concentrations in an antigen-antibody ratio of 8 : 1 (9.8-5000 ng of human aggregated IgG equivalent per ml). We found that the assay also detected an immunoreactive U1-RNP antigen in Sm : anti-Sm immune complexes but did not detect SSA : anti-SSA immune complexes. The assay was preliminary applied for serum samples obtained from patients with mixed connective tissue disease (MCTD), and elevated concentrations of nRNP immune complexes were found in 3 out of 5 patients with MCTD. This assay appears to be applicable to the detection and quantification of circulating nRNP ICs in patients with MCTD.     

Affinity of fibronectin for polyclonal IgG.

Fibronectin (Fn) is an adhesive glycoprotein that plays an important role in reticuloendothelial system functioning. Fn binds several macromolecules, it is found in cryoprecipitates obtained from patients with different diseases and it interacts in vitro with immune complexes. The interaction between Fn and polyclonal IgG was characterized by ligand affinity chromatography. After IgG was modified by attachment to a solid matrix or heat aggregation, it was able to interact with either soluble or immobilized Fn. The binding was highly influenced by ionic strength and pH. The estimated affinity constants were 3.0 x 10(6)/M when soluble Fn was applied to solid phase IgG and 2.3 x 10(7)/M when aggregated IgG interacted with immobilized Fn. The interaction may be relevant in situations in which immune complexes are involved.     

Soluble FcgammaRIIa inhibits rheumatoid factor binding to immune complexes

Soluble low-affinity receptors for IgG are known to inhibit immune complex (IC)-mediated inflammation, and expression by leukocytes is elevated in several inflammatory diseases. Immunoglobulin M (IgM) rheumatoid factors (RF), anti-Fc autoantibodies, are found in autoimmune diseases, such as rheumatoid arthritis (RA), as well as in normal immune responses. This study demonstrated that soluble FcgammaRIIa inhibits the interaction of rheumatoid factors with ICs. The recombinant soluble low-affinity FcgammaR, rsFcgammaRIIa, partially inhibited (30-70%) the rate of precipitation of soluble ICs by RF-positive RA sera. This required the normal interaction of FcgammaRIIa with Fc as the effect could be abrogated with the Fab fragment of the blocking mAb IV-3. Furthermore, rsFcgammaRIIa partially inhibited (40%) the binding of a monoclonal IgM RF (RF-AN) to an IC formed by IgG2 antibody binding to an antigen-coated biosensor chip. Since RF-AN has been characterized by crystallography to bind to the CH2/CH3 interface of the IgG-Fc, and leukocyte FcgammaRIIa binds to a distinct site centred on the lower hinge, this inhibition is uncompetitive. Some inhibition (15%) of staphylococcal protein A binding to IC was also observed. As soluble FcgammaRIIa disrupts Fc:Fc interactions in IgG-ICs, we propose that this alteration of the IC also reduces the accessibility of Fc portions in the IC, resulting in the partial inhibition of ligands, particularly IgM RF, which bind Fc. We propose that the high concentrations of soluble FcgammaR found during inflammation can affect the properties of ICs and their interaction with the immune system.     

Complement-dependent inhibition of Fc receptors on human peripheral blood mononuclear cells: inhibition of the binding of aggregated IgG,soluble and particulate immune complexes

In our previous papers the complement-dependent inhibition of aggregated IgG binding to peripheral blood mononuclear cells (PBMC) preincubated with normal human sera or with C3b generated in the presence of lymphocytes was described. In this paper the complement-dependent inhibition of the binding of soluble [¹²⁵I]BSA-anti-BSA and particulate E_huA_anti-D immune complexes (ICs) is described. Nascent C3b fragments, generated either in fresh serum during activation of the classical complement pathway or from native C3 molecules by trypsin, in the presence of the cells inhibited the binding of ICs to the cells. A comparison of the complement-dependent inhibition of different Fc-receptor (FcR) ligands revealed that the fixation of aggregated IgG was more intensively inhibited than that of particulate or soluble ICs. A possible mechanism of the binding of different FcR ligands — aggregated IgG and ICs — to the cell membrane and the causes of the differences in the inhibition induced by C3b fragments fixed to the acceptor sites on PBMCs is discussed.     

Serum and Plasma Fibronectin Binds to Complement Reacted Immune Complexes Primarily via C1q

The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after incubation of the solid-phase IC with serum or plasma at conditions where Clq was also bound, whereas only minor amounts of fibronectin bound to the solid phase IC via C3b. Purified fibronectin showed a dose-dependent binding to solid-phase IC pretreated with Clq or fibronectin-depleted serum, confirming that the binding of fibronectin to IC largely occurred via Clq. Significantly smaller amounts of fibronectin were bound to solid-phase IC incubated with plasma instead of serum, despite the higher fibronectin concentration in plasma. This difference was found not to be due to a fibrinogen-fibronectin interaction in plasma. Binding of fibronectin to preformed fluid phase IC incubated with serum was demonstrated by SDS-PAGE analysis of PEG-precipitated IC.     

IgG immune complex binding to and activation of liver cells. An in vitro study with IgG immune complexes, Kupffer cells, sinusoidal endothelial cells and hepatocytes

BACKGROUND/AIMS: The aim was to study IgG immune complex (IC) binding to isolated hepatocytes, Kupffer cells (KCs) and sinusoidal endothelial cells (SECs). Further, we wished to analyze the capacity of IgG ICs to induce release of reactive oxygen metabolites by the IC-binding liver cells. METHODS: ICs were formed between (125)I-tyramine-cellobiose-labelled dinitrophenyl-conjugated human serum albumin ((125)I-TC-DNP(10)HSA) and polyclonal rabbit IgG antibodies. Binding of ICs to different rat liver cells in suspension was studied at 4 degrees C. Production of reactive oxygen metabolites was measured by luminol-enhanced chemiluminescence at 37 degrees C. RESULTS: IgG mediated binding of (125)I-TC-DNP(10)HSA to both KCs and SECs, but not to hepatocytes. The binding showed saturation kinetics and was blocked by an excess of unlabelled IgG-ICs. IgG-ICs activated KCs, but not SECs, to a chemiluminescence response. CONCLUSIONS: Both KCs and SECs bind IgG-ICs in vitro, probably via Fc receptor interaction. IgG-ICs activate KCs to produce reactive oxygen metabolites. The binding of IgG-ICs to isolated hepatocytes is small.     

Increased serum levels of IgA1-IgG immune complexes and anti-F(ab')2 antibodies in patients with primary IgA nephropathy

A solid-phase ELISA was used to detect IgA1 immune complexes (IgA1 ICs) containing IgG and IgM in 38 serum samples from 30 patients with primary IgA nephropathy (IgAN) and 14 subjects with non-IgA chronic glomerulonephritis. A jackfruit lectin, jacalin, was used as the substrate for the selective binding of human IgA1 ICs in serum PEG precipitate (7%). The presence of IgG, A and M antibodies against the F(ab')2 region of IgG was also investigated by the solid-phase ELISA. Six patients were studied during remission and relapse (fever, upper respiratory tract infection and macroheamaturia). The results showed significant increases in serum levels of IgA1 ICs (P less than 0.001) in 39.4% of the IgAN patients, IgA1-IgG ICs (P less than 0.001) in 68.4%, and IgA1-IgM ICs (P less than 0.002) in 10.5% of the patients. A significant increase in IgA1-IgG ICs was observed during relapse (P less than 0.02). Significantly high values of IgG (P less than 0.003) and IgA (P less than 0.001) antibodies directed at the F(ab')2 region of IgG were found. A significant increase in anti F(ab')2 antibodies (class IgA and IgM) was seen in the acute phase of the disease. The data suggest that an increased production of IgA1 ICs occurs in IgAN patients; ICs are mainly IgA1-IgG ICs during relapse. The presence of high serum levels of IgG and IgA antibodies against the F(ab')2 region of IgG indicates that in addition to the multiple anomalies of IgA regulation described in IgAN patients there may be further aberrances.     

Binding of immunoglobulin G to peripheral blood lymphocytes

The specific and saturable binding of FITC conjugate of aggregated goat IgG to goat peripheral blood lymphocytes was studied in PBS containing 1% BSA. The polar nature of the specific interaction of heterologous aggregated IgG, IgG monomer and its fragment F(ab'2) with the cells was studied by ELISA using the peroxidase conjugated F(ab'2) of anti-human IgG under different conditions of pH and ionic strength.     

Soluble Fcgamma receptor IIIb alters the function of polymorphonuclear neutrophils but extends their survival.

We have established that polymorphonuclear neutrophil (PMN)-binding anti-Fcgamma receptor IIIb (FcgammaRIIIb) autoantibodies (autoAb) inhibit the function of these cells but extend their survival. Here, we show that recombinant FcgammaRIIIb (rFcgammaRIIIb), as well as purified FcgammaRIIIb (pFcgammaRIIIb), deteriorated the PMN adherence and respiratory burst in a dose-dependent manner. Furthermore, rFcgammaRIIIb and pFcgammaRIIIb reduced the level of annexin V-binding PMN from 23.6 +/- 1.6 % to 6.3 +/- 1.0 and 11.0 +/- 1.0 %, respectively, while human serum albumin exerted no effects. Incubation of rFcgammaRIIIb with those autoAb binding to soluble FcgammaRIIIb resulted in the attachment of such immune complexes (IC) to the cells, thereby also delaying apoptosis (44.9 +/- 5.9 versus 18.0 +/- 2.0 % annexin V-binding PMN after 16 hours). Soluble FcgammaRIIIb, in concert with FcgammaRIIIb / anti-FcgammaRIIIb IC, produced similar effects in that the percentage of annexin V-binding PMN declined to 16.0 +/-1.9 %. It was thus suggested that FcgammaRIIIb / anti-FcgammaRIIIb IC inserted the Fc region of their IgG into the membrane FcgammaRIIIb. Such an interpretation is consistent with our finding that, whereas aggregated IgG and anti-FcgammaRIIIb monoclonal Ab prevented membrane FcgammaRIIIb / IC interaction, neither soluble FcgammaRIIIb, nor anti-cgammaRII did so. We conclude that the function and the life span of PMN are influenced synergistically by soluble FcgammaRIIIb and anti-FcgammaRIIIb autoAb.     

Current situation and problems in the estimation of circulating immune complexes]

Some methods employing murine monoclonal antibodies have been developed for the estimation of circulating immune complexes (ICs). In the assays using monoclonal antibodies to C1q and C3d, ICs attached by reaction of C1q or C3d with the corresponding antibodies are detected by enzyme-labelled anti-IgG antibody. The murine monoclonal rheumatoid factor (RF) of IgG class is employed in the assay for detection of ICs. ICs reacted with the RF on the solid phase are further detected by the reaction with the second anti-IgG antibody labelled with the enzyme. The anti-C1q antibody in the sera as well as ICs produces positive reactions in the solid phase C1q assay, the assays using monoclonal antibodies are recommended for use in the detection of circulating ICs. In the pretreatment of serum samples, heating at 56 degrees C induces aggregation of IgG to produce a positive reaction by these sensitive assays, and the addition of EDTA-Na2 increases free C1q detached from C1 to induce increased binding to IgG. Reactions of aggregated IgG with RF and C1q in the fluid phase inhibit the following binding of monoclonal RF and anti-C1q antibody on the solid phase. Sera of patients with SLE were examined for CH50, anti-DNA antibody and ICs. The levels of ICs determined by the anti-C1q and C3d antibody assay did not correlate with other parameters. Positivity of ICs was unexpectedly lower in SLE sera. To evaluate the significance of the estimation of ICs, more data must be analyzed by these methods.     

Identification of a fibronectin binding protein from Streptococcus mutans

The interaction of viridans streptococci with components of the extracellular matrix (ECM) plays an important role in the pathogenesis of infective endocarditis. We have identified a surface protein of Streptococcus mutans which binds the ECM constituent fibronectin (Fn). Initially, we found that S. mutans could adsorb soluble Fn in plasma, but with lower efficiency than Streptococcus pyogenes. In addition, S. mutans could bind immobilized Fn in a dose-dependent manner when tested using an enzyme-linked immunosorbent assay. Crude extracts of cell wall-associated proteins or extracellular proteins from S. mutans MT8148 specifically bound Fn through a protein with the molecular mass of ca. 130 kDa, as detected by far-Western immunoblotting. The candidate Fn binding protein (FBP-130) was purified to near homogeneity by using Fn coupled Sepharose 4B affinity column chromatography. A rabbit polyclonal antibody against FBP-130 reacted specifically with a protein of molecular mass of ca. 130 kDa in both cell wall and extracellular fractions, and the abundance of FBP was higher in the former than in the latter fractions. The purified FBP bound specifically to immobilized Fn, whereas the binding of soluble Fn to coated FBP could only be detected in the presence of high concentrations of Fn. The purified FBP, as well as anti-FBP immunoglobulin G, inhibited the adherence of S. mutans to immobilized Fn and endothelial cells (ECV304) in a dose-dependent manner. These results demonstrated that FBP-130 mediated the adherence of S. mutans specifically to Fn and endothelial cells in vitro. The characteristics of S. mutans and FBP-130 in binding Fn confirmed that viridans streptococci adopt different strategies in their interaction with ECM.     

C3b binding immune complexes and immunoconglutinins in human sera. Detection by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) designed to measure autoantibodies against C3b (immunoconglutinins: IK) also detects immune complexes (IC). Solid phase C3b, in addition to binding IK of IgG, IgM and IgA classes, bound aggregated human IgG, IgA and aggregated immunologically purified anti-tetanus toxoid antibodies as well as model complexes of tetanus toxoid-human anti-toxoid. Significant C3b binding IgG, IgM and IgA activities were seen in the sera of 20 SLE patients but not in sera from healthy blood donors. Ultracentrifugation analysis of two SLE sera revealed C3b binding IgG and IgA activities in both light (7S) and heavy (11S) fractions. This indicates simultaneous presence of IK and IC in these sera. On the basis of the known relationship between IK and IC formation we suggest that the solid phase C3b ELISA may be of value in evaluating immune reactions in patients.     

Non-complement-dependent adsorption of soluble immune complexes to human red cells

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.     

Mannan binding lectin and its interaction with immunoglobulins in health and in disease

In humans there are five classes of immunoglobulins (Igs), IgG, IgM, IgA, IgE and IgD, all of which are glycoproteins. The Igs are the major secretory product of the adaptive immune system, and they bind to antigens via variable sequences on their Fab regions. The binding to antigen results in neutralization or agglutination of bound material and also initiates effector functions via the Fc regions, such as opsonisation and activation of the classical complement pathway through binding of C1q. Mannan binding lectin (MBL), the 'recognition' molecule of the lectin pathway of complement activation, is homologous in structure to C1q, and binds in a calcium-dependent manner to terminal mannose and GlcNAc residues which have been identified on the oligosaccharides N-linked to the Igs. MBL binds agalactosylated glycoforms of IgG (IgG-G0), polymeric forms of IgA and certain glycoforms of IgM which have a high incidence of GlcNAc-terminating glycans. This interaction provides a route of clearance of immune complexes from the serum, and a mechanism of complement activation to Ig-coated pathogens. In disease, MBL contributes to inflammation in Rheumatoid Arthritis, a condition in which serum IgG-G0 concentrations can increase significantly compared to healthy individuals. MBL has recently been demonstrated to bind Ig in the B cell receptor complex which expresses abnormal variable region glycosylation, in follicular lymphoma.     

Critical aspects of immune complex assays employing polyethylene glycol

Treatment of artificial immune complexes (ICs) with 2.5% polyethylene glycol (PEG) — conditions under which C1q-binding activity is routinely measured in the fluid phase — produced marked changes in molecular size as determined by Sepharose 6B chromatography. The effect of PEG on the C1q-binding capacity of ICs, was therefore investigated using a solid phase (SP) system.

PEG enhanced the binding of aggregated human gammaglobulin (AHG) and artificial ICs to SP-C1q and, in reverse experiments, also increased the binding of C1q to SP-AHG. The degree of enhancement varied according to the Ag: Ab ratio employed; the binding of ICs formed in moderate Ab excess was only modestly enhanced but that of complexes formed at slight Ab excess, equivalence and Ag excess was markedly elevated. The profile of PEG-induced enhancement of binding paralleled that of similar ICs in the C1q fluid phase system, suggesting that C1q binding in the latter may be influenced by PEG. However, the C1q-binding activity of in vivo-formed ICs seemed to be relatively unaffected by PEG since enhanced binding was comparable in control and pathological sera.

The results indicate that PEG causes cross-linking and aggregation of ICs (and possibly other serum proteins) which may alter their biological activity and hence influence the results of IC assays that employ this agent.     

The effects of various drugs and immune complexes on the function of alveolar macrophages from patients with collagen-vascular diseases with interstitial pneumonia

Alveolar macrophages (AM) taken from sixteen patients with collagen-vascular diseases with interstitial pneumonia (CVD-IP+) were cultured with prednisolone (PSL), prostaglandin E2 (PGE2), colchicine (Colch), D-penicillamine or aggregated IgG (agg-IgG). The culture supernatants were tested on the fibroblast proliferation function and the amounts of fibronectin (Fn) and interleukin 1 (IL-1). The fibroblast proliferation was suppressed significantly by the supernatants of AM cultured with PGE2. The Fn production by AM was significantly suppressed by PGE2 and Colch. The IL-1 production by AM was not suppressed by these drugs, but was enhanced by Colch. In five patients with CVD-IP+, the fibroblast proliferation of AM supernatant was enhanced by agg-IgG. In these five patients, Fn and IL-1 production had a tendency to be increased by agg-IgG. The ratio of immune complexes (IC) in broncho alveolar lavage fluids to those in sera was significantly higher in patients with CVD-IP+ than in patients with CVD without IP suggesting a local production of IC in the lung in the patients with CVD-IP+.     

Binding of human C-reactive protein (CRP) to plasma fibronectin occurs via the phosphorylcholine-binding site

Human C-reactive protein (CRP) is an acute phase reactant that is selectively deposited at sites of tissue damage. CRP binds with high affinity to purified plasma fibronectin (Fn) when the Fn is immobilized on a surface or matrix via either specific IgG antibody or by gelatin. The CRP to Fn binding is saturable at a molar ratio of CRP/Fn of approximately 9 with a Kd = 1.47 x 10(-7) M and requires Ca2+. The binding site on Fn for CRP was localized to the C-terminal portion by using monoclonal antibodies (mAbs) to Fn as competitive inhibitors of CRP. The binding involves the phosphorylcholine (PC)-binding site of CRP since the addition of PC inhibits binding to Fn and those mAbs to CRP that bind at or near the PC-binding site selectively inhibit the CRP to Fn binding. In addition the mouse IgA myeloma protein TEPC-15, which is specific for PC, also competes with CRP for binding sites on Fn. A mAb to the mouse PC-binding idiotype T-15, which also reacts with the PC-binding site of CRP, inhibits the binding of CRP to Fn. The findings suggest that CRP may play a role in the formation of the extracellular matrix needed for tissue repair. The CRP-Fn interaction may be one of the explanations for the observation of selective deposition of CRP at sites of tissue injury.     

Scatter factor binds to thrombospondin and other extracellular matrix components.

Scatter factor (SF) is an angiogenic growth factor that stimulates motility and invasion of carcinoma cells. SF is present in the extracellular matrix (ECM) of breast cancers, where it might act to promote tumor cell invasion and angiogenesis. To investigate how SF is incorporated into the ECM, we studied the binding of SF to various ECM components using a solid-phase binding assay based on the SF enzyme-linked immunosorbent assay. We found that SF binds to a variety of ECM molecules, with different binding capacities. The highest SF binding capacities were observed for thrombospondin-1 (TSP-1), fibronectin (Fn), and heparan sulfate proteoglycan, although SF did not bind to albumin. Mature two-chain SF and precursor single-chain SF bound approximately equally well to TSP-1 and Fn. Moreover, two SF alpha-chain peptides (NK1 and NK2) both bound to TSP-1 and Fn, suggesting that the whole SF molecule is not required for binding. Based on binding competition assays, TSP-1 exhibited higher affinity for SF than did nine other ECM molecules, including Fn and heparan sulfate proteoglycan. Although heparin in solution potently inhibited the binding of SF to TSP-1-coated surfaces, even very high concentrations of heparin could not elute SF already bound to TSP-1. SF binding was modulated by binding interactions among ECM molecules (TSP-1-Fn, TSP-1-collagen I, and Fn-collagen I), suggesting that the matrix capacity to bind SF depends upon its exact composition. SF bound in a dose-dependent fashion to ECMs secreted by three human breast carcinoma cell lines. Binding of SF to matrices from all three cell lines was significantly inhibited by preincubation of the matrices with antibodies against TSP-1, whereas antibodies against several other ECM components were less effective or ineffective in inhibiting SF binding. In addition, TSP-1 markedly inhibited chemotaxis of microvascular endothelial cells toward SF and SF-induced angiogenesis in the rat cornea neovascularization assay. Our findings suggest that 1) SF interacts with a variety of ECM components, 2) high affinity SF-TSP-1 interactions may mediate the binding of SF to the breast cancer matrix, and 3) the SF-TSP-1 interaction may contribute to modulation of angiogenesis. Possible implications of these findings for tumor angiogenesis are discussed.     

Influence of the electric charge of the antigen and the immune complex (IC) lattice on the IC activation of human complement

In order to understand the mechanism of complement (C) activation by immune complexes (ICs), the anti-complementary effect of ICs containing cationized antigens was compared in vitro to that using ICs formed by native antigens. ICs were prepared with affinity-purified rabbit polyclonal IgG antibovine serum albumin (BSA) antibody and either native BSA (isoelectric point 4.2) or BSA rendered cationic by treatment with ethylenediamine (isoelectric point 9.4). Native and cationized antigens were characterized by isoelectric focusing. ICs containing anti-BSA IgG or F(ab')2, formed either at equivalence or in excess of native or cationized antigen, were submitted to ultracentrifugation in a sucrose gradient for mesh size determination. The anti-complementary effect of ICs was evaluated by kinetic determination of haemolytic activity of human serum on haemolysin-sensitized sheep red blood cells. In conditions of antigen excess, the ICs formed by cationized BSA were significantly more efficient in activating human complement than those formed by native antigen. This higher activity was dependent on cationized antigen complexed with complete antibody molecules, as non-complexed cationized BSA or ICs prepared with F(ab')2 fragments were inactive under the same experimental conditions. Furthermore, this difference did not depend on the mesh size of the immune complexes. Our results suggest that the balance between antigen, antibody and C may be of importance in vivo for the onset and course of infections and other pathological processes involving IC formation. ICs containing cationized antigens should be proven of value in experimental models for studies on the regulation of C activation.     

Biosynthesis of J-chain in human lymphoid cells producing immunoglobulins of various isotypes

The relationship between synthesis, secretion, and subcellular localization of J-chain, IgM, IgA, and IgG was investigated in cultures of PWM-stimulated human PBL and in lymphoblastoid cell lines. Cells were examined for surface, cytoplasmic, and secreted immunoglobulins (Igs) and J-chain by immunofluorescence and radioimmunoassay (RIA). By these techniques, J-chain was detected in cells that produce polymeric or monomeric Igs. In PWM-stimulated PBL the synthesis of J-chain paralleled the production of Igs. In both PWM-stimulated (for 2 days) and unstimulated PBL, equal proportions of free and disulfide-linked J-chain were found. Increased amounts of intracellular J-chain were produced at later stages in PWM-stimulated PBL and J-chain occurred mostly in a free form. In tissue culture fluids, J-chain was not secreted in a free form but was always disulfide-linked to polymeric Igs. In lymphoblastoid cell lines, J-chain was present in a disulfide-linked form in IgM and IGA producers, but in IgG cells and in an IgM cell line (DAUDI) that did not secrete IgM but expressed it on the cell membrane, intracellular J-chain was present in free form. Although various proportions of polymeric and monomeric IgA were seen in culture fluids from IgA-secreting cell lines, intracellular IgA occurred mostly in a monomeric form. Further studies revealed that the ability to produce polymers was not equally distributed among all cells and might vary according to their content of J-chain and stage of maturation. Subcellular fractionation and subsequent analyses for J-chain and Ig in PWM-stimulated PBL and in IgM or IgG-producing cell lines revealed that these proteins were associated with fractions that contained ribosomes, cell sap, and low molecular weight RNA. In lysates of IgG and J-chain producing cells grown in the presence of 3H-labeled amino acids, intracellular J-chain was not disulfide-linked to IgG.