Detection of nucleosome particles in serum and plasma from patients with systemic lupus erythematosus using monoclonal antibody 4H7.

OBJECTIVE: To develop a monoclonal antibody reagent that would react with nucleosomes but not directly with constituent double stranded DNA (dsDNA) or with histones. METHODS: Mice were immunized with highly purified chicken mononucleosomes and hybridomas employed to produce Mab that did not react with dsDNA or histones but still showed reactivity with nucleosomes. RESULTS: Murine monoclonal IgG antibody 4H7, generated from a mouse immunized with highly purified chicken erythrocyte nucleosomes, showed no direct ELISA reactivity with either dsDNA or isolated histones or with Sm and RNP antigens or combinations of any of these components. Mab 4H7 did show strong ELISA reactivity for chicken erythrocyte and calf thymus nucleosomes as well as for human leukocyte nucleosomes. The Mab did show strong ELISA reactions with peptides 1-25 of histone H2B and 1-21 of H3, which correspond to sequences known to be located at the surface of nucleosomes. We then measured relative serum levels of 4H7 reactive nucleosome antigen in 140 patients with systemic lupus erythematosus (SLE) in parallel with 50 non-SLE patients with other types of connective tissue disease and 92 healthy subjects. Occasional low levels of serum nucleosomal antigen were seen in 4 of 92 controls, but many patients with SLE (66/140) showed marked elevations of serum nucleosomal antigen. No difference was observed when serum or plasma samples were studied. A marked correlation (R = 0.401, p < 0.0001) was noted when disease activity score (SLEDAI) was plotted against optical density value measured with 4H7 in ELISA. Further, the levels of circulating nucleosomes were raised in SLE patients with very active central nervous system and renal involvement. CONCLUSION: Presence of nucleosome related antigen in sera from patients with SLE may provide insight into the sequence of disease related antigenic stimuli in active SLE.

Characterization of human tumor-infiltrating lymphocytes expanded in hollow-fiber bioreactors for immunotherapy of cancer.

We attempted to grow tumor-infiltrating lymphocytes (TIL) from 34 fresh tumors of eight different histologies using flasks for the initiation phase and hollow fiber bioreactors to expand TIL to therapeutic numbers. Overall success rate was 76% (26/34) including melanoma (9/14, 64%) and renal cell carcinoma (11/11, 100%). The mean number of days required to reach successful initiation (1 x 10(9) TIL) for all tumor types was 29 +/- 16 days (mean +/- S.D.). Therapeutic doses of TIL required an average of 88 +/- 23 days (initiation plus expansion) with an average TIL number of 3.2 x 10(10) +/- 2.8 x 10(10). TIL phenotype was predominantly CD4+ in 53% (16/30) and CD8+ in 47% (14/30), renal cell carcinoma samples accounted for 12/14 of the predominantly CD8+ TIL. Cells bearing the natural killer (NK) phenotype represented only 0-7% of TIL while LAK phenotype represented 0-68% (mean 11 +/- 15%); LAK was the predominant phenotype in one patient with kidney cancer. Cytotoxicity tests showed consistent NK and LAK activity in addition to cytolysis of autologous tumor. Autologous tumor cell restricted cytolysis was noted for three TIL cultures. The overall success rate and characteristics of TIL were similar to our results with TIL expanded in semi-permeable plastic bags. This work confirms that hollow-fiber bioreactors are a suitable alternative to semi-permeable bags and roller bottle systems for the expansion of human TIL for therapeutic use in cancer patients.

Characterization of cancer cell lines established from two human metastatic breast cancers.

Cell lines are valuable resources for the study of the malignancy and potential therapy of human breast cancer. A major problem with adapting fresh breast tumor specimens to grow in vitro is contamination by fibroblasts. Previously, we have reported a technique to overcome this problem (Nayak, S. K; Dillman, R. O. Clin. Biotechnol. 3:237-242; 1991). We have recently established two new breast cancer cell lines, HH315 and HH375, that were derived from abdominal and supraclavicular lymph node metastases from two patients. They were characterized by (1) growth kinetics; (2) staining with monoclonal antibodies (MoAbs) to cytokeratin-19, epithelial membrane antigen (EMA), anticarcinoembryonic antigen (CEA), breast cancer antigen 1 (BRST-1), breast cancer antigen 2 (BRST-2), Her2/neu, and p53; (3) expression of domains of urinary plasminogen activator (uPA), neural cell adhesion molecule (NCAM), and haptoglobin (Hp) (Harvey et al., 1997); and (4) karyotypic analysis. Growth kinetic studies showed that doubling times for both lines ranged from 48 to 96 h. These two cell lines were found to have characteristics of the metastatic breast cancer cells. Both lines stained positive with MoAbs to cytokeratin-19 and EMA, thus confirming their epithelial origin. They also strongly reacted with the pan-breast carcinoma MoAbs BRST-1 and BRST-2, and carcinoembryonic CEA MoAb. Both cell lines overexpressed the oncogene proteins Her2/neu and p53. The tumor cells were negative for estrogen and progesterone receptors. HH315 cells were poorly differentiated, whereas the HH375 cells exhibited adenocarcinoma morphology. Both cell lines showed intense cell surface and some cytoplasmic staining for uPA, NCAM, and Hp domains, which is a characteristic of malignant neoplasms (Harvey et al., 1997). The HH375 cell line showed two cell types, of which 60% were hyperdiploids with 60-70 chromosomes and 5-10 marker chromosomes. The remaining cells were polyploid with more than 200 chromosomes. Cell line HH315 consisted of only a polyploid population. These cell lines may be useful in breast cancer research.

Determination of primary amino acid sequence and unique three-dimensional structure of WGH1, a monoclonal human IgM antibody with anti-PR3 specificity.

Transformed B cells making monoclonal IgM-lambda anti-PR3 antibody WGH1 from a patient with Wegener's granulomatosis were used to prepare mRNA and synthesize cDNA. PCR primers for human micro and lambda chains were then employed to amplify heavy- and light-chain V-regions followed by cloning into pCR2-1 vector and sequencing. Molecular modeling of VH regions employed knowledge-based homology modeling to obtain minimum energy conformation. The VH sequence was subgroup III with marked overall homology to VH1.9III. The VHCDR3 region of WGH1 was unique, consisting of 21 amino acid residues which included seven tyrosines as well as three negatively charged aspartic acid residues. The VL region was subgroup II with a negatively charged glutamic acid at position 100 in CDR3. Molecular modeling of VH revealed a major conformational difference in the shape of CDR3 compared with other antibodies for which three-dimensional structures have been determined. Monoclonal antibody WGH1 reacting with PR3 (a highly positively charged molecule) shows a unique reactive cassette within VHCDR3 with a number of negatively charged aspartic acid residues. WGH1 VHCDR3 contains a loop which shows a major projection not usually recorded in other previously studied antibody molecules.

Urinary loss of immunoglobulin G anti-F(ab)2 and anti-DNA antibody in systemic lupus erythematosus nephritis.

The objective of this study was to determine whether the low levels of serum immunoglobulin G (IgG) anti-F(ab)2 seen in some patients with active systemic lupus erythematosus (SLE) were directly related to the deposition of antibody with this specificity in the kidney or alternatively to the urinary loss of IgG anti-F(ab)2. Serum Levels of IgG anti-F(ab)2, anti-tetanus toxoid, and anti-ds DNA antibody were measured in parallel with urinary excretion of these same 3 antibodies in 28 patients with SLE nephritis and in 28 control patients with other forms of chronic kidney disease. Low levels of both serum IgG anti-F(ab)2 or anti-tetanus antibody appeared to correlate with increased levels of urinary loss of these same antibodies in some patients with SLE and in control subjects with kidney disease. However, urinary loss could not account for low serum levels of either IgG antibody in many subjects. Quantitative 24-hour urinary losses of IgG anti-F(ab)2 and anti-DNA were much higher in patients with SLE than in control subjects with kidney disease (P < .05), whereas amounts of IgG urinary loss of anti-tetanus were similar in patients with SLE and in control subjects. In nearly 1 third of SLE nephritis patients, 13% to 53% of total excreted urinary IgG showed anti-DNA enzyme-linked-immunosorbent assay reactivity. Urinary IgG in many patients with SLE showed both anti-DNA and anti-F(ab)2 reactivity, but dual anti-DNA/F(ab)2 specificity was more pronounced in affinity-isolated serum IgG anti-DNA or anti-F(ab)2 than in excreted urinary IgG molecules. The affinity of urinary IgG for either DNA or F(ab)2 was much lower than the same antibody activities measured either in serum or in kidney biopsy eluates. When the relative affinity of anti-DNA antibody in serum, urine, and kidney biopsy eluate was measured in parallel, the highest affinity antibody was found in kidney biopsy eluates, followed by serum antibody with urine antibody affinity showing the lowest values. These findings suggest a relative concentration of the highest affinity, doubly reactive IgG anti-DNA/F(ab)2 in SLE kidney tissues during SLE nephritis and implicate this process as an important factor in ongoing tissue damage.

Antigenic determinants reacting with rheumatoid factor: epitopes with different primary sequences share similar conformation.

Polyclonal or monoclonal human IgM rheumatoid factors (RF) react with eight antigenic sites on the CH3 IgG domain, four sites on CH2 and two on human beta 2-microglobulin. All 14 of these RF-reactive epitopes are linear 7-11 amino acid peptides with different primary sequence. We questioned whether RF reactivity with such a variety of epitopes showing no obvious sequence homology might result from conformational similarities shared by various RF-reactive regions. Strong support for this concept was obtained using rabbit antisera as well as mouse mAbs to individual CH3, CH2 or beta 2m RF-reactive peptides. Major cross-reactivity was demonstrated between most of the 14 different CH3, CH2, or beta 2m RF-reactive peptides using individual anti-epitope antibodies. Molecular modelling studies of these peptides showed striking similarities in three-dimensional shape among many RF-reactive peptides. Main-chain atoms rather than side chains seemed to contribute most directly to conformational similarity. Molecular simulation studies on control peptides showed no conformational similarities with RF-reactive peptides. Our studies indicate that autoantibodies such as RF recognize main-chain conformations of reactive epitopes and react with a number of antigenic determinants of quite different primary sequence but similar main chain conformations.

Affinity columns containing anti-DNA Id+ human myeloma proteins adsorb human epibodies from intravenous gamma globulin.

OBJECTIVE: To study eluates of intravenous gamma globulin (IVGG) prepared from affinity columns of human cationic IgG myeloma proteins bearing anti-DNA idiotype (Id) markers 16/6, F4, 3I, and 8.12 for possible anti-Id (combining site) blocking activity. METHODS: Anti-DNA idiotypic antibody activity was studied in 3 preparations of IVGG containing high, medium, and low levels of IgG anti-F(ab')2, and in 4 other commercial IVGG preparations. Affinity-purified IgG anti-DNA (APAD) from systemic lupus erythematosus (SLE) patients was biotinylated, and binding to DNA coated on enzyme-linked immunosorbent assay plates was used to measure anti-DNA antibody activity. IVGG was adsorbed to Sepharose 4B affinity columns linked to a panel of cationic human IgG myeloma proteins positive for anti-DNA Id markers 16/6, F4, 3I, and 8.12. Material adsorbing to such columns was eluted at low pH (2.5) and after neutralization, tested for its ability to inhibit biotinylated APAD reacting with DNA. RESULTS: Only 0.05-0.9% of IVGGs bound firmly to Id affinity columns. These IVGGs were then eluted, using pH 2.5 glycine-saline and eluates neutralized to pH 7.4. Column flowthrough and eluate fractions were compared for their ability to block SLE APAD reacting with DNA. Significant inhibition of SLE APAD combining sites was observed with eluates from anti-DNA Id affinity columns; however, no correlation between IVGG anti-F(ab')2 activity and true anti-Id blocking of APAD was apparent. No residual anti-Id activity remained in column flowthrough fractions. No anti-Id blocking activity was recorded for IVGG eluates from human cationic myeloma columns devoid of the 4 anti-DNA Id markers. DNase treatment of IVGG or Id column eluates did not affect anti-Id blocking activity. Thus, all detectable anti-DNA idiotypic antibody capable of blocking SLE anti-DNA combining sites bound to Id+ affinity columns. Column eluates also showed some relative concentration of IgG anti-DNA activity, which was of lower affinity for DNA than antibodies also present in eluates which blocked anti-DNA combining sites. CONCLUSION: The presence of both anti-DNA and antiidiotypic (anti-combining site) activity in human anti-DNA Id column eluates indicates that epibodies from IVGG are relatively concentrated when this strategy is used. This approach may lead to a new strategy for treatment of SLE nephritis.

Cross-reactivity of human IgG anti-F(ab')2 antibody with DNA and other nuclear antigens.

OBJECTIVE: To characterize immunologic specificity and possible antiidiotype activity of IgG anti-F(ab')2 in normal subjects as well as in patients with active and inactive systemic lupus erythematosus (SLE). METHODS: IgG anti-F(ab')2 and anti-double-stranded DNA (anti-dsDNA) were affinity isolated from immunoadsorption columns of F(ab')2 and dsDNA linked to Sepharose 4B. Affinity-purified IgG anti-F(ab')2 (APAF) and affinity-isolated IgG anti-dsDNA (APAD) were tested by enzyme-linked immunosorbent assay (ELISA) for other cross-reacting specificities including anti-Sm, anti-Sm/RNP, and anti- Crithidia binding. Anti-DNA specificity of APAF and APAD was assayed by S1 nuclease treatment of heat-denatured DNA. Rabbit antiidiotypic antisera were prepared by immunization with APAF and APAD from normal subjects and SLE patients and absorption with insolubilized human Cohn fraction II (Fr II). VL and VH regions of 5 monoclonal IgM antibodies with anti-F(ab')2/anti-DNA specificity generated by Epstein-Barr virus B cell stimulation were sequenced by polymerase chain reaction and characterized for VH and VL subgroup. APAF and APAD were also examined by high-resolution electron microscopy for possible ring forms indicative of antiidiotypic V-region interactions. RESULTS: APAF from normal subjects, representing 0.08-0.18% of serum IgG, showed striking relative concentrations of both anti-F(ab')2 and anti-DNA, as well as anti-Sm and anti-Sm/RNP ELISA reactivity. Both APAF and APAD reacting with F(ab')2 or dsDNA on the ELISA plate could be cross-inhibited by F(ab')2 or DNA in solution. Anti-DNA reactivity in normal APAF and APAD was much more sensitive to S1 nuclease treatment than similar fractions from SLE patients. Neither APAF nor APAD from controls produced positive antinuclear immunofluorescence or positive Crithidia staining, whereas these were strongly positive using SLE APAF and APAD. Absorbed rabbit antisera against normal or SLE APAF and APAD showed strong ELISA reactivity against both APAF and APAD, but no residual reactivity with normal Fr II. VL and VH sequencing of monoclonal human IgM antibodies showing both anti-F(ab')2 and anti-DNA reactivity showed relative VH3, V kappa 1 or VH1, V kappa 3 restriction. No evidence of ring forms or V-region "kissing" dimers was obtained when normal or SLE APAD or APAF was examined by high-resolution electron microscopy. CONCLUSION: IgG anti-F(ab')2 in both normal subjects and SLE patients represents a polyreactive Ig subfraction with concomitant anti-DNA, anti-Sm, and anti-Sm/RNP specificities. Anti-DNA reactivity in SLE is qualitatively different from that in normal APAD and APAF since normal APAD and APAF anti-DNA is much more sensitive to S1 nuclease digestion of denatured dsDNA. APAF and APAD share distinct V-region antigens which may be related to prominent VH3 or VH1 antigenic components. No evidence for in vivo complexing of anti-DNA and anti-F(ab')2 as ring forms or antiidiotype-IgG complexes was observed during ultrastructural studies. In both normal individuals and SLE patients, APAF may represent a small polyreactive IgG subfraction which also contains antinuclear and anti-DNA specificities.

Benign monoclonal gammopathy with IgG anti-DNA, anti-Sm and anti-F(ab')2 activity.

OBJECTIVE: To study anti-DNA idiotypic markers and anti-DNA activity in human monoclonal immunoglobulin proteins. METHODS: Seventy human IgG M-components intentionally selected for cationic electrophoretic characteristics were studied for F4 and 31 anti-DNA idiotypic markers and anti-DNA as well as anti-F(ab')2 antibody activity. RESULTS: Eight of 70 M-components showed significant anti-DNA activity. In two both anti-DNA and anti-F(ab')2 activity occurred together. One IgG-2 kappa M-component showed extremely high anti-ds DNA, anti-Sm, anti-F(ab')2 and anti-Sm/ RNP ELISA activity. Cross inhibition studies showed that each reactive antigen inhibited the other. N-terminal V-region sequencing showed the VH3, VK3 subgroup. Anti-idiotypic rabbit antibody produced against this M-component showed strong reactivity with affinity purified IgG anti-DNA and anti-F(ab')2 from most SLE patients and normal subjects. CONCLUSION: Monoclonal human immunoglobulins may contain multiple autoantibody specificities including anti-DNA; anti-Sm, anti-Sm/RNP, and anti-F(ab')2. Many antibodies with these specificities share common V-region antigens. Such relationships could contribute to idiotypic immune regulation and control.

Autoantibodies as chameleons.

Autoantibody determinations are frequently used by rheumatologists to establish the diagnosis or assess follow up clinical status in patients with connective tissue diseases. Such autoantibodies are often presumed to have harmful effects, particularly since some such as anti-native DNA or anti-Ro have frequently been related to tissue damage or to functional impairments. However, there are many other autoantibodies which react with antigenic components of normal autologous tissues which have not been demonstrated to have self-damaging or harmful effects. Some of these autoantibodies may actually represent natural built-in mechanisms of feed-back inhibition, serving to modulate normal physiologic function. Autoantibodies may be compared to chameleons since their function or quality is often judged by the company they keep or by their anatomical localization. Since many autoantibodies to intra-cellular products seem to react with active sites of important biologic molecules, they may provide us with a much sharper image of a number of natural cellular functions.

Cationic myeloma M-components frequently show cross-reacting anti-DNA, Anti-F(ab')2 and anti-nucleosome specificities.

134 cationic human IgG myeloma proteins were studied for expression of anti-DNA Idiotypic markers. 64 were studied for 16/6, F4, 3I, and 8.12, and 70 for expression of F4 and 3I. 31.3% showed at least one anti-DNA Id marker and many cationic myelomas were also positive for anti-DNA ELISA reactivity as well as anti-F(ab')2. Five M-components showed anti-nucleosome reactivity and one without detectable anti-DNA Id markers showed very strong anti-nucleosome antibody which was also inhibited by DNA and Sm antigens. Anti-idiotypic antisera produced either against Id(+) anti-DNA reactive M components or F(ab')2 fragments of affinity purified SLE IgG anti-DNA showed preferential cross-reactive idiotype reactivity between Id(+) anti-DNA reactive M components. Our findings indicate that human IgG monoclonal proteins positive for several common anti-DNA Ids and possessing anti-DNA ELISA reactivity, can serve as models for SLE Id marker antigens and as a source to prepare anti-Ids from IVIG.

Shared V-region antigens and cross-reacting specificities of human IgG anti-F(ab')2 and anti-DNA antibodies.

Affinity-isolated IgG anti-F(ab')2 (APAF) and anti-DNA (APAD) from normal subjects as well as patients with systemic lupus erythematosus (SLE) were studied for overlapping antibody specificities as well as shared V-region antigens using rabbit antisera prepared against F(ab')2 fragments of SLE IgG APAD or IgG APAF from normal individuals. When absorbed free of antibody reacting with normal human IgG, both types of rabbit anti-Ids showed cross-reacting antigenic determinants shared by APAF and APAD from both normals and SLE patients. Immunodepletion of APAD or APAF IgG fractions using immunoabsorbents composed of such rabbit anti-SLE F(ab')2 APAD or anti-normal IgG APAF antibodies produced substantial decrements (40-90%) in ELISA anti-F(ab')2, antiSm, anti-Sm/RNP, and anti-DNA reactivity. Parallel decrements in human anti-DNA Id markers F4 and 3I were also recorded after immunoadsorbent depletions of SLE APADs. Absorption of rabbit IgG anti-SLE APAD F(ab')2 antibody with DNA-Sepharose produced a relative increment in ELISA anti-DNA reactivity but a marked parallel decrement in reactivity for the original SLE APAD immunogen. Eluates from DNA-Sepharose contained rabbit epibody showing moderate anti-DNA affinity and high-affinity anti-APAD idiotypic activity. This epibody fraction showed relative concentration of rabbit IgG antibodies capable of blocking combining sites of SLE IgG APAD reacting with DNA on the ELISA plate. Our findings indicate a previously unexpected close antigenic and specificity overlap between human IgG anti-F(ab')2 and anti-DNA antibodies. Moreover, they emphasize a surprising relative infrequency of anti-DNA idiotypic antibodies capable of blocking combining sites in animals immunized with SLE F(ab')2 APAD. This finding may relate to an intrinsic defect of anti-Id regulation in SLE.

IgM rheumatoid factors react with human class I HLA molecules.

Half of 30 human polyclonal IgM rheumatoid factors (RF) showed positive ELISA reactions with affinity-isolated human class I molecules (A2 and B7). Positive RF reactivity with isolated class I heavy chains indicated that anti-class I RF interaction did not merely reflect RF anti-beta 2m specificity. Cross-reactions between antigenic determinants on human IgG, class I molecules, and beta 2m reacting with RF could be demonstrated by ELISA inhibition. When gene products of A2 alpha 2 exons 2, 3, and 4 were synthesized as overlapping heptamers on polypropylene pins, six RF-reactive epitopes within solvent accessible class I HLA regions were identified: EPRAPWI, QEGPEYW, QTHRVDL (second A2 exon), EQLRAYL, GTCVEWL, and WLRRYLE (third A2 exon). Glycine-alanine substitution for each residue within these RF-reactive sites identified R48, W51, E55, Y107, R108, W147, Q155, and E172 as immunodominant residues for RF reactivity. Many of these RF-reactive class I regions also showed positive ELISA reactions with monoclonal human IgM RFs derived from rheumatoid arthritis synovial B cells. Whole serum from patients with rheumatoid arthritis showed a spectrum of IgM, IgA, and IgG Abs that adsorbed to and could be eluted from monomeric IgG or HLA A2 affinity columns. Normal serum similarly analyzed showed only trace reactions with IgG, A2, or beta 2m. Flow cytometry using RF incubated with A2 cell lines showed definite immunofluorescence reactivity with cell membranes not observed with normal non-RF IgM. Reactivity of human IgM RF with determinants on class I molecules may reflect antigenic overlap within several members of Ig gene superfamily products.

Immunity in the connective tissue diseases. The humoral side of the coin.

Clinicians who care for patients with various connective tissue diseases frequently employ measurements of autoantibodies such as rheumatoid factors (RFs), anti-Sm antibodies, or anti-neutrophil cytoplasmic antibodies (cANCA) as a method to follow patients. Although the primary specificity of RFs appears to be directed against the Fc portion (C gamma 3 and C gamma 2 domains) of IgG, epitope mapping studies have now also demonstrated that many RFs also react with linear regions on beta 2-microglobulin and Class I HLA molecules. Cross reacting regions of IgG, beta 2m, and HLA Class I frequently show immunodominant tyrosines, trytophanes, valines, leucines, glutamic acids, aspartic acids, and threonines. Immunodominant linear epitopes on Sm antigen may be limited to regions expressing the PPPGMRPP or PPPGIRGP motifs. A number of linear regions of Proteinase 3 reacting with IgG antibodies in the sera of patients with Wegener's granulomatosis have now been identified. However, affinity purified rabbit antibodies to two of these major PR3 antigenic sties (ATVQLPQ and RVGAHDP) linked to Sepharose to form affinity columns, absorbed equal amounts of a mixture of many serum proteins from both Wegener's patients and normal controls. Continued study of this interface between autoantibody production, disease, and normal immune modulation is necessary.

IgG anti-F(ab')2 antibodies from SLE patients react with immunodominant residues in kappa CDRs, but show reduced C kappa region reactivity.

We studied reactivity of affinity-purified human IgG anti-F(ab')2 antibodies for antigenic determinants on kappa light chains. Variable (VL) and constant (CL) regions of a human kappa I light chain (EU) were studied for enzyme-linked immunosorbent assay reactivity with monoclonal anti-k antibodies and human IgG antiF(ab')2 using overlapping heptamers of primary sequence synthesized as peptides on polypropylene pins. Polyclonal IgG anti-F(ab')2 antibodies isolated by affinity chromatography from the serum of patients with systemic lupus erythematosus (SLE) were found to react primarily with CDR1- and CDR3-related peptides. The most reactive residues included IL29, TRY32, LEU33, ALA34, GLU90, TYR91, and ASN 92. No striking difference in overall V-region anti-F(ab')2 reactivity profiles was noted when 10 IgG anti-F(ab')2 preparations from normal subjects and SLE patients were compared. In contrast, however, when tested against overlapping C kappa heptamer sequences, the reactivity in SLE-associated anti-F(ab')2 antibodies was markedly decreased. We report that this difference may reflect a defect in anti-idiotypic control mechanisms in SLE.

Molecular localization of human IgG anti-F(ab')2 reactivity with variable- and constant-region lambda light-chain epitopes.

Human IgG antibodies reacting with antigenic determinants on F(ab')2 fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially lambda type, we synthesized constant (C)lambda- and variable (V)lambda-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab')2-reactive epitopes on human lambda light chains. ELISA reactivity of affinity-purified anti-F(ab')2 antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for C lambda and V lambda subgroup-related determinants, was tested using the overlapping 7-mers of human lambda light-chain sequence. The patterns of reactivity against C lambda-related peptides were similar in both normal and SLE-derived anti-F(ab')2 antibodies. However, reactivity profiles against V lambda-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab')2 autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab')2 antibodies was noted for particular amino acid V lambda complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant V lambda CDR residues.

CDR molecular localization of possible anti-idiotypic anti-DNA antibodies in normal subjects, patients with SLE, and SLE first-degree relatives.

Patients with active systemic lupus erythematosus (SLE) with disease worsening or severe flares frequently show very low levels of serum immunoglobulin G (IgG) anti-F(ab')2 antibody. Anti-F(ab')2 antibody probably represents a polyclonal collection of generic anti-idiotypic antibodies involved in immune homeostasis. We synthesized the entire variable regions of the heavy and light chains (VH and VL) of two monoclonal anti-DNA antibodies, V88 and 2A4, as overlapping 7-mers on small polypropylene pins and tested these linear segments of anti-DNA V-regions for reactivity against serum samples from 10 normal subjects with high serum IgG anti-F(ab')2, 11 normal subjects with low anti-F(ab')2, 5 patients with SLE with active uncontrolled disease, 3 patients with SLE in remission, and 8 unaffected normal first-degree SLE relatives. VH and VL regions of a human monoclonal IgG anti-rabies antibody were also tested as a control. Concordant IgG antibody reacting with the same complementarity-determining regions (CDRs) was arbitrarily scored as indicative of the presence of anti-idiotypic antibody in test serum samples. Among normal subjects with either high or low serum anti-F(ab')2 levels, 10% to 21% showed strong concordant anti-CDR reactions with either the monoclonal anti-DNA or the control monoclonal anti-rabies V-region sequences. However, all patients with active SLE showed no detectable anti-CDR-reactive antibody. Patients with SLE in remission often showed return of strong concordant anti-CDR antibody. Normal unaffected SLE relatives also showed high levels of anti-CDR reactivity for both monoclonal anti-DNA and anti-rabies antibody sequences.

Active systemic lupus erythematosus is associated with depletion of the natural generic anti-idiotype (anti-F(ab')2) system.

OBJECTIVE: To study the relationship of serum IgG anti-F(ab')2 and clinical disease activity in 108 patients with systemic lupus erythematosus (SLE) and to determine whether low serum anti-F(ab')2 with active renal disease is accompanied by deposition of anti-F(ab')2 in renal immune complex lesions. METHODS: We studied 108 patients with definite SLE over a 5 yr period and assayed serum anti-F(ab')2 levels in relation to degree of clinical disease activity. Renal biopsy eluates of 26 patients with SLE were examined by enzyme linked immunosorbent assay (ELISA) for relative amounts of IgG, anti-DNA, and IgG anti-F(ab')2. RESULTS: Active SLE was strongly associated with low serum anti-F(ab')2. SLE renal biopsy eluates frequently contained high levels of IgG and IgG anti-DNA and lower, but definite, IgG anti-F(ab')2 activity. When specific activity of IgG anti-DNA and IgG anti-F(ab')2 was compared between kidney biopsy eluates and concomitant serum, marked relative renal concentration was found for both anti-DNA (19-fold) and anti-F(ab')2 (74-fold). Some biopsy eluates contained IgG antibodies bearing apparent double specificity for both DNA and F(ab')2. CONCLUSION: Active SLE is often associated with low serum anti-F(ab')2. Relative enrichment over specific activity in serum of both IgG anti-F(ab')2 and anti-DNA in SLE kidney biopsy eluates may indicate participation of both reactants in the glomerular disease process. Low serum anti-F(ab')2 in active SLE may reflect down modulation of failure of idiotypic control mechanisms associated with disease progression.

Rheumatoid-factor-reactive sites on CH3 established by overlapping 7-mer peptide epitope analysis.

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis and six monoclonal IgM RF were isolated from monomeric IgG affinity columns and studied for their reactivity with the entire CH3 domain of IgG synthesized as overlapping 7-mers using a pin-ELISA assay. All ten polyclonal IgM RF showed similar profiles of reactivity which included peptides with solvent accessible residues PREPQVY (residues 343-349), PQVYTLP (residues 346-352), TLPPRSE (350-356), DGSFFLY (401-407), WQQGNVF (417-423), CSVMHEG (425-430), EGLHNHY (430-436) and KSLSLSP (439-446) of the CH3 domain. Substitution of a neutral glycine or alanine for each residue within these RF-reactive epitopes indicated that tyrosine at position 349, prolines at 343, 346 and 352, glutamine 347, valine 348, threonine 350, leucine 351, arginine 354, aspartic acid 401, tyrosine 407, serine 426, histidine 429, leucine 432, tyrosine 436 and lysine 439 represented important single amino acids within CH3 for RF reactivity. Regions of CH3 primary sequence with and without the single allotype-specific amino acid substitutions of glycine for alanine 431 (Gmx) or aspartic acid for glutamic acid (356) and leucine for methionine (358) (Gma) often showed considerable differences in reactivity with individual polyclonal and monoclonal RF. However, these differences in RF reactivity did not correlate with the individual anti-Gm RF specificity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a much more restricted spectrum of reactive CH3 epitopes. 7-mer peptides representing RF-reactive sites on CH3 preincubated with polyclonal IgM RF showed strong inhibition (55-66%) of RF binding to whole IgG on the ELISA plate. These studies indicate that it is possible to define portions of the IgG CH3 domain participating in the reaction with IgM RF using reactive epitope-mapping with sequential linear peptides derived from the primary IgG CH3 sequence.

Studies of protein A and herpes simplex virus-1 induced Fc gamma-binding specificities. Different binding patterns for IgG3 from Caucasian and Oriental subjects.

Herpes simplex virus type 1 (HSV-1) expresses a receptor that binds the Fc portion of IgG. This HSV-1 Fc gamma-binding protein is, like protein A of Staphylococcus aureus, known to bind human IgG1, IgG2 and IgG4 but not IgG3 subclasses. However, IgG3 with the allotype Gm(s+)(t+), prominent in the Oriental population, reacts with protein A. This prompted us to investigate the reactivity of Oriental IgG3 monoclonal myeloma proteins of various allotypes with the HSV-1 Fc gamma-binding protein. Of seven Oriental IgG3 myeloma proteins with allotypes Gm(s+)(t+)(u-)(b+)(g-), Gm(s-)(t-)(u+)(b+)(g-) and Gm(s-)(t-)(u+)(b-)(g+), all reacted with the HSV-1 Fc gamma-binding protein. This was in contrast to negative reactions obtained with three IgG3 myeloma proteins of Caucasian origin with Gm(b+)(g-) or Gm(b-)(g+) phenotypes. The same binding pattern, i.e. binding of IgG3 of Oriental but not of Caucasian origin, was found with protein A. The binding of the monoclonal Oriental IgG3 proteins was again independent of the G3m phenotype. These findings support the concept that the HSV-1 Fc gamma-binding protein A have a similar binding site on the IgG molecule. All monoclonal IgG3 proteins derived from Oriental subjects with or without histidine at position 435 bound to HSV Fc gamma-binding protein. This suggests that Oriental IgG3 myeloma proteins with Gm(s-)(t-) phenotypes have additional critical amino acid residue substitutions important for HSV Fc gamma binding different from those already known.