Heat-aggregated Human IgG Research Articles

免疫複合体検出法 (Clq-microplate ELISA) における Clq と可溶性熱会合 IgG との結合への単体 IgG の阻害作用

免疫複合体検出法 (Clq-microplate ELISA) における Clq と可溶性熱会合 IgG との結合への単体 IgG の阻害作用

Systemic lupus erythematosus monocytes are less responsive to interleukin‐10 in the presence of immune complexes

Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by autoantibody production and immune complex deposition. The level of interleukin-10 (IL-10), predominantly an antiinflammatory cytokine, is paradoxically elevated in patients with SLE. The aim of this study was to examine the hypothesis that the antiinflammatory function of IL-10 is impaired in monocytes from patients with SLE with long-term exposure to immune complexes. CD14+ monocytes were isolated from healthy donors and patients with SLE. Cultured CD14+ cells were treated with heat-aggregated human IgG (325 μg/ml) in the presence or absence of IL-10 (20 ng/ml). To study gene expression, RNA was extracted 3 hours after treatment. To study cytokine production, supernatants were harvested after 8 hours. To study IL-10 signaling, cell lysates were obtained from CD14+ cells treated with human IgG (325 μg/ml) for 1 hour followed by IL-10 (20 ng/ml) treatment for 10 minutes. Western blot analysis was used to assess STAT-3 phosphorylation. All experiments were performed in pairs. When stimulated with human IgG, SLE monocytes produced more tumor necrosis factor α (TNFα) and IL-6 than did control cells. The suppressive effect of IL-10 on human IgG-induced TNFα and IL-6 production was lower in SLE monocytes compared with control monocytes, although IL-10 receptor expression was similar in SLE and control monocytes. Human IgG suppressed IL-10 receptor expression and altered IL-10 signaling in control monocytes. Like SLE monocytes, interferon-α (IFNα)-primed control monocytes stimulated with human IgG were also less responsive to IL-10. Human IgG and IFNα modulate IL-10 function. In SLE monocytes, which are considered to be IFNα primed and persistently exposed to immune complexes, responses to IL-10 are abnormal, limiting the antiinflammatory effect of this cytokine.

In vitro comparison of the complement‐scavenging capacity of different intravenous immunoglobulin preparations

Complement inhibition is considered important in the mechanism of action of intravenous immunoglobulin (IVIG) in a number of inflammatory and autoimmune disorders. The capacity of different IVIG preparations to 'scavenge' activated C3 and thereby inhibit complement activation was assessed by a new in vitro assay. Diluted human serum as a complement source, with or without addition of different concentrations of IVIG, was incubated in microtitre plates coated with heat-aggregated human IgG. Complement scavenging was measured by detecting reduced C3 binding and determining fluid phase C3b-IgG complex formation. Complement activation induced by the IVIG preparations was measured as C5a formation. All IVIG preparations exhibited a dose-dependent inhibition of C3b deposition, correlating strongly with binding of C3b to fluid-phase IgG, but the extent of complement scavenging varied considerably between different IVIG preparations. At an IVIG concentration of 0.9 mg/ml, the inhibition of C3b deposition ranged from 72 +/- 16% to 22 +/- 4.1%. The reduction of C3b deposition on the complement-activating surface was not due to IVIG-induced complement activation in the fluid phase, as shown by the low C5a formation in the presence of serum. In vitro analysis allows comparison of the complement-inhibitory properties of IVIG preparations. The extent of complement scavenging varies between the products.

Insoluble immune complexes are most effective at triggering IL-10 production in human monocytes and synergize with TLR ligands and C5a

Purpose In systemic lupus erythematosus (SLE), a disease of immune complex (IC) deposition, interleukin-10 (IL-10) is thought to promote B-lymphocyte hyperactivity and autoantibody production. Both ICs and Toll-like receptor (TLR) ligands have been shown to stimulate the production of IL-10 by human monocytes. Using an in vitro model, we studied how IC solubility, complement activation products, and TLR ligands could affect IL-10 production by human monocytes stimulated with ICs. Methods Human monocytes were stimulated with soluble or insoluble heat-aggregated human IgG with or without TLR ligands or C5a. Cytokine levels in cell culture supernatants were measured by ELISA. To study cytokine signaling, cell lysates were analyzed by Western blot for total or tyrosine-phosphorylated STAT3. Results Insoluble ICs were most effective at stimulating production of IL-10, and costimulation LPS enhanced synthesis of IL-10. In addition, stimulation with insoluble ICs together with C5a enhanced the production of IL-10 by 2–4 fold in either the presence or absence of TLR ligands. Increased STAT3 phosphorylation correlated temporally with enhanced IL-10 production and was reduced by an IL-10 receptor blocking antibody, suggesting that IL-10 was responsible for observed STAT3 phosphorylation. Conclusions Because the immune deposits of SLE are, by definition, insoluble; and because IL-10 is thought to be important for B-cell hyperactivity and autoantibody production, these observations provide a critical link, bridging current views of B-cell hyperactivity with the early concept that SLE may arise from defective clearance of immune complexes.

Signaling Pathways in HIT Immune Complex Stimulation of Monocyte Tissue Factor.

In heparin-induced thrombocytopenia (HIT), platelet-fibrin thrombi form in vivo due to generation of thrombin initiated by intravascular tissue factor (TF). Stimulation of monocytic cells in vitro by the HIT immune complex (IC), consisting of heparin/PF4 complexes and IgG anti-hep/PF4 antibodies, triggers TF expression. The HIT IC binds to Fcγ receptors on monocytic cells to initiate a signal cascade which results in new TF transcription. Though much is known about monocyte TF transcription as a consequence of LPS stimulation of TLR4, the mechanism by which HIT IC engagement of FcγR leads to TF transcription is unknown. Transcription of pro-inflammatory and anti-inflammatory cytokines as a consequence of IC engagement of FcγRs on monocytes has been reported to involve the ras/Erk and/or PI3K/Akt pathways downstream of syk. We tested the hypothesis that HIT IC- FcγR engagement signals via a PI3K/Akt pathway to trigger TF transcription. We created a model HIT IC consisting of an optimal ratio of heparin and recombinant human PF4, bound by well-characterized monoclonal anti-hep/hPF4 antibody KKO. Using human monocytic cell line THP-1, we observed that the HIT IC induction of new TF mRNA, assessed by qRT-PCR after treatment for 2 h at 37°C, was reduced approximately 80% by pretreatment of the cells with PI3K inhibitor wortmannin (100 nM; n = 3, p < 0.05). In addition, the same effect was observed with another IC, heat-aggregated human IgG, namely 80% inhibition of stimulated TF expression by wortmannin (pretreatment with 100 nM, 37°C, 30 min, n = 4, p< 0.05). Of note, the LPS-mediated increase in TF expression was enhanced over 2-fold (1 ug/ml, 2 h, p <0.05) by the same wortmannin pretreatment, in direct contrast to the result with IC stimulation. In preliminary studies with Akt inhibitor SH5, we have observed ~80% inhibition of IC-stimulated TF expression. We have verified the effects of PI3K inhibition of IC-stimulation of TF (75–80% inhibition by wortmannin pretreatment) by using primary peritoneal macrophages derived from FcγRIIa-transgenic mice, which express a similar FcγR pattern as human cells and our HIT mouse model. Immunoblot analysis of phospho-Akt using anti-Ser473 Akt antibody(generously provided by Donna Woulfe, PhD, Thomas Jefferson University) verified the phosphorylation of Akt by HIT IC treatment and marked reduction when PI3K was inhibited. In summary, HIT IC stimulation of monocytic cell TF involves a PI3K/Akt pathway in a way clearly distinct from LPS-stimulated TF expression. Further studies are in progress to elucidate the upstream activators and downstream effectors, the role of other signaling pathways and the identity of the responsible transcription factor complexes.

OxLDL immune complexes activate complement and induce cytokine production by MonoMac 6 cells and human macrophages

Oxidized low density lipoprotein (OxLDL) is immunogenic and induces autoimmune responses in humans. OxLDL antibodies are predominantly of the proinflammatory IgG1 and IgG3 isotypes. We tested the capacity of immune complexes prepared with copper-oxidized human LDL and affinity chromatography-purified human OxLDL antibodies [OxLDL-immune complexes (ICs)] to activate complement and to induce cytokine release by MonoMac 6 (MM6) cells and by primary human macrophages. The levels of C4d and C3a were significantly higher in human serum incubated with OxLDL-ICs than after incubation with OxLDL or OxLDL antibody, indicating complement activation by the classical pathway. MM6 cells and primary human macrophages were incubated with OxLDL-ICs, with or without prior conditioning with interferon-gamma. After 18 h of incubation, both MM6 cells and primary human macrophages released significantly higher levels of proinflammatory cytokines after incubation with OxLDL-ICs than after incubation with OxLDL or with OxLDL antibody, both in primed and unprimed cells. OxLDL-ICs were more potent activators of MM6 cells than keyhole limpet hemocyanin-ICs. Blocking Fc gamma receptor I (FcgammaRI) with monomeric IgG1 significantly depressed the response of MM6 cells to OxLDL-ICs. In conclusion, human OxLDL-ICs have proinflammatory properties, as reflected by their capacity to activate the classical pathway of complement and to induce proinflammatory cytokine release from MM6 cells and primary human macrophages.

A novel immunoadsorbent for rheumatoid arthritis therapy--preparation and efficacy evaluation.

To develop a novel immunoadsorbent for rheumatoid arthritis (RA) therapy. A RA immunoadsorbent was developed by binding heat-aggregated human IgG(HAHIgG) to porous agar gel beads. Its adsorption capacity for rheumatoid factors (RFs), storage stability and blood compatibility were evaluated. The coupling yield of HAHIgG on the carrier was 6.0 mg/g wet gel. Saturation adsorption capacity of the adsorbent for IgMRF, IgGRF and IgARF were 3400, 2240 and 2400 IU/g, respectively. The adsorbent can be stored at 4 degrees C for three months without significant variance in its activity. Its fine permeability and hemocompatibility were demonstrated by extracorporeal hemoperfusion on rabbits. HAHIgG/agar gel is a safe and effective immunoadsorbent for RA therapy, its potential clinical use is promising in the future.

Heat-Aggregated Human IgG As Immunosorbent for Rheumatoid Arthritis

In order to selectively remove pathogenic rheumatoid factors (RF) from plasma of rheumatoid arthritis (RA) patients, a new immunosorbent was developed by immobilizing heat-aggregated human IgG (HAH IgG) on epichlorohydrin-activated agar gel. Different parameters influencing ligand coupling were studied, i.e., ligand concentration, reaction time, temperature and pH. The maximum amount of HAH IgG immobilized on the carrier was 2.7 mg/ml. Adsorption capacity was evaluated by ELISA technique. After incubation of 3.0 ml of RA plasma with 1.0 ml of the adsorbent, the RF IgM, IgG, and IgA were removed by 88%, 74%, and 50%, respectively. The decrease of total immunoglobulins and albumin were less than 7% and 9%. In vitro, perfusion of 10.0 ml plasma at 37 degrees C through 3.0 ml of adsorbent, at a flow rate of 5.0 ml/min for 60 min, resulted in the removal of 49% of RF IgM, 72% of RF IgG and 58% of RF IgA. No leakage of ligand and no decline in adsorption capacity and mechanical strength were observed after steam-sterilization.

Immune complexes are potent inhibitors of interleukin-12 secretion by human monocytes.

We have studied the effect of immune complexes (IC) on interleukin (IL)-12 secretion by human monocytes in vitro. Two experimental models of IC were used. IC formed of tetanus toxoid and polyclonal anti-tetanus toxoid antiserum as well as heat-aggregated human serum IgG almost completely inhibited IL-12 (p70 and p40) secretion induced by interferon-gamma and lipopolysaccharide in human blood-derived monocytes. Neutralizing anti-IL-10 antibodies plus indomethacin restored IL-12 secretion in the presence of IC to a high extent, indicating that IL-10 and prostaglandin (PG) partially mediate the IC-induced inhibition of IL-12 secretion. However, neutralization of tumor necrosis factor (TNF)-alpha by specific antibodies also incompletely restored IL-12 secretion. Indeed, monocytes secrete high levels of TNF-alpha upon stimulation by IC. We found that exogenously added TNF-alpha caused a profound inhibition of monocytic IL-12 secretion in the absence of IC, again mediated via the induction of IL-10 and PG. In summary, IC inhibit IL-12 secretion via TNF-alpha-induced IL-10 and PG synthesis. We conclude that IC, typically appearing in the course of chronic inflammatory processes, may influence the balance between Th1 and Th2 responses and may thus contribute to a deprivation of cell-mediated immune responses.

FcγRII on Human B Cells Can Mediate Enhanced Antigen Presentation

The ability of FcγR on monocytes and macrophages to enhance presentation of Ab-bound Ag has been well established. In contrast, studies suggest that FcγRII on B cells functions primarily to suppress B cell responses. We found that presentation of tetanus toxoid (TT) by human EBV-transformed B cells was enhanced by 100- to 1000-fold, when TT was targeted to FcγRII on B cells by using a conjugate consisting of TT covalently linked to anti-human FcγRII mAb 41H16. This enhanced presentation could be blocked by mAb 41H16, heat-aggregated human IgG, or anti-MHC Class II mAb. Similarly, multimeric immune complexes composed of TT and anti-TT mAb, SA13, and 9F12 also enhanced presentation of TT. Furthermore, FcγRII on purified human peripheral blood B cells could also enhance Ag presentation. These studies provide the first clear evidence that FcγRII can participate in enhanced Ag presentation by human B cells, and suggest that FcγRII has the potential to play a stimulatory role in human B cell responses to Ag–Ab complexes.

IgG-Fc receptors in stratum granulosum: an immunological defence in human skin?

Receptors for the Fc-part of IgG (Fc tau R) in stratum granulosum of normal human skin were examined using cryosections and indirect immunofluorescence staining with 1) soluble immune complexes and 2) monoclonal antibodies (MoAbs) against different types of Fc tau R, i.e. 32.2 (anti-Fc tau I-CD64), IV.3 (anti-Fc tau RII-CD32) and Leu 11b (anti-Fc tau RIII-CD16). The immune complexes gave staining corresponding to stratum granulosum in sections from all skin specimens. Inhibition experiments showed that pre-incubation of the sections with monomeric and heat-aggregated human IgG, periodic acid and formaldehyde inhibited the immune complex binding. F(ab')2 containing immune complexes did not bind to the skin sections. The MoAb 32.2 gave granular and Leu 11b linear staining corresponding to stratum granulosum. In addition, both IC, 32.2 and Leu 11b gave weaker staining of keratinocytes in other parts of the epidermis. IV.3 stained epidermal Langerhans' cells and were unreactive with other epidermal cells. Indirect immunofluorescence staining with MoAbs against IgG subclasses showed the presence of all IgG subclasses in stratum granulosum. The results show that granulosum cells express both high- and low-affinity IgG receptors and in vivo bound IgG. The data point to a role for stratum granulosum in cutaneous immunity.

Chimaeric anti‐CD4 monoclonal antibody cross‐linked by monocyte Fcγ receptor mediates apoptosis of human CD4 lymphocytes

Previous studies have shown that murine anti-CD4 monoclonal antibody, cross-linked by rabbit anti-mouse immunoglobulin, could mediate apoptosis of murine CD4+ lymphocytes when they were stimulated by T cell receptor antibody. In this study, we have shown that the murine anti-CD4 monoclonal antibody, OKT4, can induce apoptosis in human CD4+ T cells stimulated by the recall antigen tuberculin purified protein derivative (PPD) only when cross-linked by rabbit anti-mouse immunoglobulin. The chimeric anti-CD4 monoclonal antibody, cM-T412 whose Fc fragment is human, was able to cause apoptosis without cross-linking by a second antibody. Similarly, abolition of PPD-induced proliferation of peripheral blood mononuclear cells by cM-T412 did not require cross-linking with rabbit anti-human immunoglobulin. Inhibition of proliferation by cM-T412 could be reduced by pre-treating monocytes with heat-aggregated human IgG. This suggested that monocyte Fc gamma receptors might be cross-linking the human Fc of cM-T412. Propidium iodide staining together with immunofluorescence showed that the apoptotic cells were indeed CD4+ lymphocytes. It is proposed that during treatment with cM-T412 in autoimmune disease such as rheumatoid arthritis, cM-T412-coated CD4 T cells, when they are subsequently stimulated by the unknown arthritogenic antigen, may undergo apoptotic cell death through cross-linking of cM-T412 on Fc gamma receptor-positive cells within the joint.

An Immune Complex Selective Affinity Column Utilizing Site-Specific Attachment of Bovine Conglutinin

A simple method to isolate immune complexes by column chromatography is described. The immune complex affinity column was constructed by the site-specific attachment of bovine conglutinin to agarose. Covalent attachment of conglutinin to agarose was achieved via hydrazone chemistry, which reacts aidehydes of oxidized oligosaccharides in the collagenous domain of conglutinin with hydrazide functional groups in the solid support. The constructed conglutinin affinity column captured complement-fixed model immune complexes of heat-aggregated human IgG and more classical complexes of chicken ovalbumin-antichicken ovalbumin. Neither the nonfixed immune complexes nor their individual components were retained by the column. The captured material was specifically eluted with EDTA without the use of low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and protein sequence analysis of the eluates revealed the presence of the expected individual components, verifying that both antibody and antigen used to prepare the soluble immune complexes wore recovered from the conglutinin column. The advantages of this approach over traditional methods of immune complex isolation and characterization are discussed.

High molecular weight Non-Immunoglobulin Salivary Agglutinins (NIA) bind C1q globular heads and have the potential to activate the first complement component

Non-Immunoglobulin Salivary Agglutinins (NIA) which directly bind to microbes [including HIV] were studied for their potential to activate the first complement component (C1). It was determined that NIA had the same specific activity as heat aggregated IgG in binding to C1q and in activating C1. In order to determine the region of Clq which bound to NIA, C1q globular heads and C1q stems (collagen-like regions) were prepared and separated via a Western blot procedure. NIA bound principally to the globular heads of C1q and weakly to the collagen-like stem region. NIA were also studied for their potential to activate native C1 in normal human serum. Heat-aggregated IgG and cardiolipin served as positive controls. It was observed that incubation of isolated NIA with fresh normal human serum resulted in the formation of sodium dodecyl sulfate (SDS)-irreversible complexes of activated C1r-C1 inhibitor and activated C1s-C1 inhibitor and in activated C1s mediated C4 conversion. This indicated that isolated NIA had the potential to directly and effectively mediate classical complement pathway activation. Preincubation of NIA with C1q, blocked NIA mediated C1r and Cls activation and C4 conversion. The concn of NIA required to activate C1r and C1s was similar to that of heat-aggregated human IgG. In kinetic ELISA, NIA or aggregated IgG (positive controls) were first immobilized on microtiter plates, blocked with gelatin then incubated with fresh human serum as a source of complement. Depositions of C4b, C3b and iC3b substantiated that the complement system was effectively activated by immobilized NIA. The optimal relative NaC1 concn for C4b deposition was 0.11 M. While pre-incubation of NIA with C1q blocked the subsequent C1 fixing potential of NIA, pre-incubation of NIA with rgp160 [HIV-1] or fibronectin did not interfere with the potential of NIA to fix C1.

Automated Turbidimetry of Rheumatoid Factor without Heat Inactivation of Serum

We describe an improved turbidimetric procedure for rheumatoid factor measurement in human serum. Heat-aggregated human IgG is used as antigen. Interference produced by the complement component C1q, which required a previous heat pretreatment of sera, is avoided by the addition of the chemical inhibitor, poly(vinyl sulphonic acid). This inhibitor eliminates C1q interference without affecting the attachment of rheumatoid factor to the Fc part of IgG, thus permitting full automation of the assay. After studying the reactivity of different heat-aggregated IgG preparations, we optimized the procedure for preparing the antigen in order to attain maximal reactivity: IgG was heated at 63 degrees C until the absorbance at 340 nm of a 1/10 dilution of the antigen lay between 0.65 and 0.95 absorbance units. The study of antigen stability showed a maximum aggregation two weeks after preparation. The antigen was then stable and could be used in the assay. The standard curve extends from 30 to 500 x 10(3) IU/l. Intra- and inter-assay CV were less than 5.5%. Relative analytical sensitivity and specificity were 98.3% and 95.2%, respectively. The results agreed well with those obtained by the non-improved turbidimetric procedure (r = 0.996) and with nephelometry values on the Behring nephelometer analyser (r = 0.964). The correlation with an enzyme immunoassay was also good (r = 0.923).

Detection of circulating immune complexes with polyethylene glycol precipitation F(ab′) 2 anti-C 3 ELISA

A polyethylene glycol (PEG) precipitation F(ab′) 2 anti-C 3 ELISA for the detection of complement-fixing IgG circulating immune complexes (CIC) is described. For this assay, test sera were treated with 3.5% PEG and then measured with F(ab′) 2 anti-C 3 ELISA. The lower detection limit was 4 μg/ml of heat aggregated human IgG (HAHG). Intra-assay coefficient of variation (CV) was 4.9–8.3%. High levels of CIC are found in the sera of patients with systemic lupus erythematosus (SLE), hepatitis B and stomach cancer.

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3 + leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3 + leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3 + LGL leukemia were cultured with 1 μg/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3 + leukemic LGL.

Mesangial macromolecular uptake in captopril-treated uninephrectomized rats

Captopril, an angiotensin II-converting enzyme inhibitor, ameliorates the renal mesangial lesions associated with subtotal nephrectomy, a process associated with increased mesangial macromolecular flux and injury. In the present study uninephrectomized rats with proteinuria and focal glomerular sclerosis had increased mesangial heat-aggregated human IgG (AHIgG) uptake. However, uninephrectomized rats treated daily with captopril, which failed to develop either glomerular lesions or proteinuria, also had significantly elevated mesangial AHIgG levels. Our results suggest that increased mesangial macromolecular flux may occur independent of altered glomerular permselectivity changes and proteinuria and appears to be related to glomerular hyperfiltration rather than glomerular hypertension. Further, glomerular mesangial sclerosis may not be the direct result of increased mesangial macromolecular flux.

The location of binding sites on C1q for DNA.

Previous studies have suggested that C1q reacts with DNA via both the globular region of C1q (GR) and the collagen-like region of C1q (CLR). In this study, the binding of dsDNA and ssDNA to GR and CLR was quantitated by a solid-phase assay. Both dsDNA and ssDNA bound to the GR and CLR of C1q in an ionic strength-dependent manner. Under physiologic salt concentrations, however, dsDNA and ssDNA bound preferentially to CLR and not to GR. The binding of dsDNA to C1q was not affected by heat inactivation of C1q or its exposure to pH 4.45, which abolished the binding of heat-aggregated human IgG (AHG) with C1q. The preincubation of the solid-phase C1q with AHG did not decrease the binding of dsDNA or ssDNA to the solid-phase C1q. These results indicate that the major sites for binding DNA to C1q are located in the CLR of C1q and are not overlapping with those for AHG or immune complexes.

Immunoglobulin Class and Subclass Profile of the Ro/SS-A Autoantibody Response

We have developed an enzyme-linked immunosorbent assay (ELISA) for autoantibody to Ro/SS-A antigen (anti-Ro/SS-A) in order to more fully characterize the autoimmune response that occurs to this antigen in patients with subacute cutaneous lupus erythematosus (SCLE). The microtiter plate-immobilized, biochemically purified Ro/SS-A antigen reacted with anti-Ro/SS-A antibody, but not with other closely related specificities (anti-La/SS-B, anti-SM, anti-U1-RNP) or normal sera. The optimal pH of antigen-antibody reaction in this ELISA was 7.2. The binding of sera containing anti-Ro/SS-A was inhibited 80% by preincubation with the same amount of Ro/SS-A antigen used for coating the plate. Although 11 of the 14 (79%) SCLE sera studied had precipitating anti-Ro/SS-A antibody by immunodiffusion, 13 (93%) sera had abnormally elevated IgG, IgA, or IgM ELISA binding levels. A good correlation between IgG anti-Ro/SS-A ELISA binding levels and immunodiffusion titers was observed (r - 0.8588, p less than or equal to 0.001) suggesting that IgG is the major anti-Ro/SS-A antibody class detected by double immunodiffusion, Sera with a combination of high rheumatoid factor levels (latex 3+ or higher) and high anti-Ro/SS-A titers (1:8 or higher in immunodiffusion) tended to give an abnormally high IgM anti-Ro/SS-A ELISA binding levels. After rheumatoid factor activity was removed by absorption with heat-aggregated human IgG, a 50% decrease in IgM anti-Ro/SS-A ELISA binding was noted. On the other hand, absorption of rheumatoid factor-negative sera that contained high IgM anti-Ro/SS-A binding activity did not significantly decrease ELISA binding levels. Prednisone and 6-azathioprine reduced the level of IgG anti-Ro/SS-A autoantibody in sera of treated SCLE patients by 50%. The IgG subclass profile of anti-Ro/SS-A autoantibody was analyzed by using mouse monoclonal antibodies specific for the 4 human IgG subclasses. Of anti-Ro/SS-A positive SCLE sera, 91% had predominantly IgG1 subclass autoantibody. The coexistence of IgM and IgG anti-Ro/SS-A autoantibody and the predominance of the IgG1 subclass is compatible with the possibility that this autoantibody response is under T-cell control. The predominance of IgG1 in the autoimmune response to Ro/SS-A antigen in SCLE patients is consistent with the hypothesis that antibody dependent cell mediated cytotoxicity could be an important immunologic effector mechanism in this disorder.