Pathogenic idiotypes of autoantibodies in autoimmunity: lessons from new experimental models of SLE

Systemic lupus erythematosus (SLE) is considered a classical autoimmune disease that involves many biological systems. Similar to other autoimmune conditions, its etiology is multifactorial entailing genetic, environmental, hormonal, and immunologic factors. In this review we demonstrate that by using a pathogenic idiotype of anti-DNA autoantibodies, it is possible to explain some of the pathogenesis and diversity of clinical and serological manifestations reported by SLE patients. The 16/6 idiotype (Id) is a representative pathogenic idiotype of anti-DNA autoantibodies. The serum titers of 16/6 Id in SLE patients correlate with clinical activity of the disease, and are deposited in afflicted tissues in SLE patients. SLE was experimentally induced in naive mice after immunization with 1 microgram of the Id. The disease is characterized clinically (proteinuria), serologically (e.g., anti-dsDNA, anti-Sm antibodies), and by pathological findings (e.g., deposition of 16/6 Id in the kidneys). The condition can be induced by other human and mouse antibodies carrying the 16/6 Id, as well as by mouse antimonoclonal-16/6 Id and by T cell lines and clones specifically reactive with 16/6 Id. There are strain-dependent differences in susceptibility to the induction of systemic lupus erythematosus (SLE). Induction of SLE is directly correlated with the ability to respond to the 16/6 idiotype (or 16/6 Id)2 by anti-Id antibody production. It is easier to induce the disease in females, and it can be modulated by manipulation of sex hormones. Being able to identify the pathogenic idiotype allowed us to generate T suppressor (Ts) cells specific for the 16/6 Id. Treatment of mice with these T cells abrogated the disease. Our studies point to the importance of pathogenic idiotypes of autoantibodies in autoimmunity, which suggests that SLE may represent a dysregulation of a functional network of idiotypes-anti-idiotypes interactions among autoreactive B cells, T helper cells, and T suppressor cells.     

Lupus serum and normal human serum contain anti-DNA antibodies with the same idiotypic marker

We have previously shown that serum from patients with active SLE contain high levels of Id-16/6 and anti-DNA antibodies. In this study we investigated whether serum Id 16/6 is related to anti-DNA antibodies. Sera from 12 patients with active SLE were absorbed individually with poly(dT) cellulose (to purify anti-DNA antibodies) and rabbit (R) anti-Id-16/6 Sepharose (to purify Id 16/6 Ig). Removal of all anti-DNA activity removed most of the Id-16/6. Conversely, removal of all Id 16/6 removed most of the anti-DNA activity. Although there was no measurable anti-DNA antibody activity in normal serum, such antibodies were isolated by absorption with poly(dT) cellulose. The eluted immunoglobulins also had Id 16/6 activity. Similarly, Id 16/6 with anti-DNA activity were isolated from normal serum by absorption with R anti-Id 16/6 Sepharose. We conclude that a large fraction of anti-DNA antibodies in SLE serum are Id-16/6+, and that most Id 16/6 immunoglobulins in lupus serum have anti-DNA activity. Our observations suggest that lupus anti-DNA antibodies result from an overproduction of autoantibodies that are present in normal people.     

Idiotypic analysis of anti-I-Ak monoclonal antibodies. III. T- and B-cell responses to anti-Ia idiotopes are not modulated by syngeneic anti-idiotypic monoclonal antibodies

To investigate whether anti-idiotypic (anti-Id) antibodies activate T cells either directly or indirectly, we examined the ability of syngeneic anti-Id monoclonal antibodies (mAbs) to regulate idiotype (Id) expression, antigen-binding antibody production, and T-cell reactivity to antigen. Our idiotypic system consists of an anti-I-A mAb that carries an infrequently expressed Id. Using three syngeneic anti-Id mAbs (Ab2), we previously defined the idiotype of the 11-5.2.1.9 (11-5) anti-I-Ak mAb. Two of these mAbs, IIID1 and IA2, recognize the same or closely related epitopes on 11-5 and cross react with two additional anti-I-Ak mAbs, 8B and 39J; the third anti-Id mAb, VC6, recognizes a distinct epitope shared by 11-5 and 8B. In the present study, BALB/c (H-2d) mice were primed with varying doses of these anti-Ids and were then boosted with C3H (H-2k) spleen cells. Among 130 such primed mice, the syngeneic anti-Ids when tested at priming doses between 10 ng and 10 micrograms were unable to induce Id production. The priming anti-Id mAbs persisted in the serum of the mice and were detectable as late as 40 days after priming. Ab1 expression was not modulated in BALB/c mice immunized with KLH-coupled Ab2, however, this immunization elicited the production of Ab3 which shared idiotypes with 11-5, 8B, and 39J. BALB/c anti-C3H alloreactive T-cell clones were also not induced by anti-Id priming, nor could they be shown to bind directly to the three Ab2 used. Nevertheless, the proliferative response of one anti-I-Ak specific T-cell clone that recognizes the same epitope as 11-5, 8B, and 39J, was inhibited by the IIID1 and IA2 Ab2. Thus, a T cell can express an idiotype shared by a B cell, but the linked recognition of an Id-associated carrier determinant(s) by an alloreactive T cell is required to elicit an anti-Id antibody response. These results favor the possibility that the activation of T cells is not dependent upon their ability to bind to anti-Id, but rather on their capacity to respond to epitopes of Id-anti-Id antigen-antibody complexes formed on B cells.     

Modulation of SLE induction in naive mice by specific T cells with suppressor activity to pathogenic anti-DNA idiotype.

T cells (CD8+) with specific suppressor activity against anti-dsDNA antibody (16/6 Id+) were generated in vitro. The cells were established from BALB/c-enriched T cells exposed in vitro to silica beads coated with the pathogenic anti-DNA idiotype, 16/6. The idiotype specificity of the suppressor cells was demonstrated by (a) specific induction of a decrease in proliferative response of T helper cell lines specific for the pathogenic idiotype (16/6 Id), when exposed to the idiotype, with no effect on T cell lines with other specificities, e.g., against human IgM or synthetic polypeptide. (b) Effectively suppressing in vitro antibody production of anti-16/6 antibody, employing 16/6-primed B cells and specific helper T cell line. The 16/6 Id-specific Ts cells were found to be MHC restricted. Weekly intravenous injections of 10(7) 16/6 Id-specific Ts cells given to BALB/c mice at different stages of experimental SLE disease prevented the clinical, serological, and pathological manifestations. This effect was characterized by decreased titers of autoantibodies (e.g., anti-DNA, anti-Sm antibodies) in the sera, by abolishment of the proteinuria, leukopenia, and the increased ESR, followed by decreased immunoglobulin deposition in the kidneys. Treating the mice with control IgM-specific T cells did not affect the above parameters. These studies demonstrate the ability to generate Ts cells specific for pathogenic idiotypes. The method might be employed therapeutically to modulate the course of autoimmune conditions.     

The role of the idiotypic network in the induction of experimental systemic lupus erythematosus

Systemic lupus erythematosus (SLE) has been induced in C3H.SW mice by their immunization with a human monoclonal anti-DNA antibody that bears a common idiotype-16/6 Id. Following immunization, high levels of murine anti-16/6 and anti-anti-16/6 antibodies were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with ssDNA, dsDNA, poly(I), poly(G), RNP, Ro, and La were also observed. The serological findings were associated with significant proteinuria, leukopenia, and elevated erythrocyte sedimentation rate. Immune complex deposition in the glomerular mesangium and sclerosis of the glomeruli were demonstrated. To study whether or not anti-idiotype antibodies are involved in the induction of the disease, a murine monoclonal antibody against the 16/6 Id was prepared and injected into C3H.SW mice. The anti-16/6 Id antibody induced experimental SLE similarly to the 16/6 Id with an accelerated kidney pathology. A study performed on different mouse strains indicated that the susceptibility to the induction of SLE by the 16/6 Id is strain dependent and directly correlates to their ability to produce anti-16/6 Id specific antibodies.     

Idiotypic and antiidiotypic T and B lymphocytes in myasthenia gravis.

The prevalence of Id and anti-Id T and B cells as measured by their reactivities with two human mAb, one antiacetylcholine receptor mAb and one anti-Id mAb, was studied in 38 patients with myasthenia gravis and in 27 healthy individuals. Id and anti-Id T cells were estimated by enumerating the numbers of cells secreting IFN-gamma in response to 10 pg/ml of the human mAb. T cell stimulation, measured as numbers of IFN-gamma-secreting cells that exceeded the mean + 2 SD of controls, was induced by the Id mAb in 78.9% of the patients and in 7.4% of the controls, whereas the anti-Id mAb-stimulated T cells in 55.3% of the patients and in 3.7% of the controls. The mean value of the Id and anti-Id-reactive T cells in the patients was 18.3/10(5) and 10.1/10(5) PBMC, respectively. B cells secreting IgM antibodies binding to the human mAb were increased in patients with myasthenia gravis compared to healthy controls. Seventy-five percent of the patients and 12% of the controls had B cells secreting IgM antibodies binding to the Id mAb, although 89% of the patients and 16% of the controls had B cells secreting IgM antibodies binding to the anti-Id mAb. The mean value of B cells secreting IgM antibodies binding to Id or anti-Id mAb in the patients were 7.4 cells/10(6) and 5.5 cells/10(6) PBMC, respectively. We conclude that Id and anti-Id T and B cells are present in myasthenia gravis. These methods allow a quantitative estimation of T and B cells with defined specificities and thus a way of mapping the repertoire of lymphocytes.     

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.     

IL-1 beta-deficient mice are resistant to induction of experimental SLE

IL-1 is one of the most pleiotropic pro-inflammatory and immunostimulatory cytokines. Overproduction of IL-1 has been shown to be involved in the pathogenicity of various autoimmune inflammatory diseases, including systemic lupus erythematosus (SLE). However, the different contributions that the IL-1 agonistic molecules make in their in vivo native milieu, IL-1beta which is mainly secreted against IL-1alpha which is mainly cell-associated, have not been established. Experimental SLE can be induced in mice by injection with monoclonal anti-DNA antibodies bearing a major idiotype designated, 16/6Id. In the present study, experimental SLE was induced in mice deficient in specific IL-1 molecules, i.e. IL-1alpha(-/-), IL-1beta(-/-), IL-1alpha/beta(-/-) (double KO) and in control BALB/c mice. Mice deficient in IL-1beta , i.e. IL-1beta(-/-) and IL-1alpha/beta(-/-) mice, developed lower levels of anti-dsDNA antibodies after immunization with 16/6Id, as compared to IL-1alpha(-/-) or control BALB/c mice. Disease manifestations were milder in mice deficient in IL-1beta expression. The representative cytokine cascade that is characteristic of overt experimental SLE was also shown to be reduced in groups of mice that lacked IL-1beta as compared to mice deficient in IL-1alpha, which is mainly cell-associated. Altogether, our results point to the importance of secretable IL-1beta, rather than cell-associated IL-1alpha, in the immunostimulatory and inflammatory phenomena that mediate the pathogenesis of experimental SLE.     

A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.     

Influence of the immunoglobulin heavy chain locus on expression of the VK1GAC light chain

The VK1GAC light chain represents the dominant V kappa structure employed in the antibody response of A/J mice to streptococcal group A carbohydrate ( GAC ). Two anti-idiotypic antisera, anti- Id5 and anti- Id20 , with specificity for the VK1GAC light chain were used to examine anti- GAC antibody responses in a series of inbred mouse strains that differ at the heavy chain constant region ( IgCH ) allotype locus. Both idiotypes were expressed in normal and immune sera from mice of most IgCH allotypes, except IgCHb (C57BL/6J) and IgCHf (CE/J). C57BL/6J mice expressed Id5 , but not Id20 , whereas CE/J mice did not express either idiotype. Testing of recombinant inbred strains between BALB/c and C57BL/6 indicated that the pattern of idiotype expression did not correlate with IgCH allotype. The C X B recombinants expressed all three idiotype patterns that were observed in the panel of inbred strains. Testing of allotype congenic mice between BALB/c and C57BL/6 showed that CB.20 and BC.8 mice were Id20 -, whereas BAB-14 mice were Id20 +, indicating that both VH and background (V kappa or regulatory) loci must be derived from BALB/c to obtain Id20 expression. The difference in the frequency of idiotype expression observed between BALB/c and BAB-14 mice indicates that the IgCH locus may exert a quantitative influence on the expression of this light chain. To examine the Id20 -, Id5 + antibodies of C57BL/6 mice, anti- GAC hybridomas were prepared. Of 16 C57BL/6-derived anti- GAC monoclonal antibodies, six were reactive with anti- Id5 and not with anti- Id20 . Isoelectric focusing of the purified kappa light chains from three of these antibodies revealed two distinct spectrotypes that co-migrated with the two known VK1GAC spectrotypes observed with A/J anti- GAC light chains. Idiotypic analysis of in vitro recombinants between the heavy and light chains of A/J and C57BL/6 monoclonal antibodies demonstrated that the C57BL/6 light chains were idiotypically similar to A/J light chains when they were free in solution or paired with A/J heavy chains. These results demonstrate that C57BL/6 mice can express a light chain that is very similar, if not identical, to the VK1GAC light chain, although the light chain is expressed in lower frequency and is paired with a distinct VH structure, which can mask expression of one of the VK1GAC idiotypes. These effects on V kappa expression map to at least three genetic loci: VH, CH, and an unlinked locus.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Characterization of syngeneic rat monoclonal antibodies to the HSN tumor using syngeneic monoclonal anti-idiotopic antibodies

Twelve syngeneic anti-idiotopic mAb (anti-idiotypic/idiotopic antibodies Ab2)) were prepared from CBH/Cbi rats immunized with one of three monoclonal anti-HSN antibodies (Ab1) (11/160, ALN/11/53, or ALN/16/53) specific for the HSN tumor. The sera of the rats used for hybridoma production and all of the monoclonal Ab2 specifically inhibited the binding to HSN of the immunizing Ab1 only. It is concluded that, in this completely syngeneic system, only the private idiotopes associated with the antibody-combining site were immunogenic. Analyses using Western blotting showed that the Ab2 bound to intact Ab1 and to isolated H chains where the intra-strand disulfide bonds remained intact. The Ab2 did not bind to L chains or to fully reduced H chains of the Ab1. It is concluded that the idiotopes expressed on the H chain were conformational. When a panel of 13 monoclonal Ab1 (including the three used for immunization) were tested for reactivity with the Ab2, three reacted specifically with their respective Ab2 and 8 gave no binding suggesting that each Ab1 had a distinct idiotypic specificity despite the fact that all the Ab1 competed with each other for binding to Ag. However, the two remaining Ab1 (ALN/9/94 and ALN/12/17) generated from different tumor-bearing rats, were found to possess the same idiotypic specificity as 11/160. A detailed analysis using seven Ab2 raised against 11/160 showed that while the idiotype of ALN/9/94 and 11/160 were very similar, that of ALN/12/17 showed some clear differences. These three Ab1 have been shown previously to bind a sequential epitope on the HSN Ag in Western blots and it is postulated that the common idiotype of these Ab1 reflects their recognition of a sequential epitope. This may also account for the relatively frequent occurrence in tumor bearer sera of antibodies with this Id.     

Human immune response to group A streptococcal carbohydrate (A-CHO). II. Antigen-independent stimulation of IgM anti-A-CHO production in purified B cells by a monoclonal anti-idiotopic antibody

The paper describes the induction by a monoclonal anti-idiotopic antibody (anti-Id mAb) of specific antibody production to group A streptococcal carbohydrate (A-CHO) in purified human B cells of several unrelated individuals. The anti-Id mAb, designated 16F498 (anti-Id498), recognizes a recurrent idiotope (Id 498) associated with the combining site of human antibodies to N-acetyl-D-glucosamine (GlcNAc), the immunodominant group of A-CHO. Id498 is expressed on IgM anti-GlcNAc antibodies but does not occur on IgG antibodies with the same specificity. It occurs also on a minor population of IgM antibodies without specificity for A-CHO. Id498 was found in 19 of 27 sera from unselected healthy donors and thus seems to be frequently expressed within the adult B cell repertoire. The in vitro induction of anti-A-CHO antibodies was analyzed in human B cells extensively depleted for T cells. Specific antibody secretion required cross-linked anti-Id which was achieved by coupling the mAb to agarose beads. No antibody secretion could be induced by soluble anti-Id (1 and 10 micrograms/ml). An optimal response required soluble T cell-derived factors which were added as a mixture of recombinant interleukin 2 with a T cell hybridoma supernatant that augments B cell growth and differentiation. Under these conditions an antigen-independent specific increase of IgM anti-A-CHO production (2.6- to 10-fold, or up to 2000 ng/ml respectively) could be induced in blood B cell populations of four of six normal individuals expressing the Id498 at serum level.     

EIevated serum level of Thioredoxin in patients with Hepatocellular Carcinoma

Thioredoxin (TRX) is known to contain an active site with aredox-active disulfide and has various biological activities. The objectiveof the present study was to investigate whether circulating TRX levels areelevated in patients with chronic hepatitis (CH) or liver cirrhosis (LC) andhepatocellular carcinoma (HCC). An anti-TRX monoclonal antibody andpolyclonal antibodies that specifically recognize TRX, were generated andused for the development of an ELlSA system to measure TRX levels in humanserum. The geometric mean and its 95% confidence interval of serumlevel of TRX in healthy volunteers was 81.75 ng/ml (74.60-89.59 ng/ml). Theserum level of TRX in LC/CH patients without HCC was 80.87 ng/ml(69.66-93.88 ng/ml). The value was not statistically different from that inserum from normal volunteers (p=0.69). In contrast, the serum level of TRXin patients with HCC was 147.35 ng/ml (125.53-1 72.96 ng/ml), which wassignificantly higher when compared with the level in serum of normalvolunteers (p<0.001) and in serum of LC/CH patients without HCC(p<0.001). In four patients with HCC, the initially high level of serum TRX(>150 ng/ml) decreased below 150 ng/ml after surgical removal of the tumor.The data reported herein revealed that patients with HCC had a significantlyelevated serum level of TRX, suggesting that measurement of serum of TRXmight be a useful clinical parameter when HCC is suspected.     

Down-regulation of stromal cell-derived factor-1alpha-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-beta secretion

Systemic lupus erythematosus (SLE) can be induced in mice by immunizing them with a monoclonal human anti-DNA Ab that expresses a major Id, designated 16/6Id. In addition, a peptide based on the sequence of the CDR 1 (hCDR1) of the 16/6Id ameliorated the clinical manifestations of SLE in experimental models. In this study we examined the effects of treating mice with human complementary-determining region 1 (hCDR1) on the subsequent chemotaxis of T cells derived from 16/6Id-primed mice. First we demonstrated elevated levels of stromal cell-derived factor-1alpha (SDF-1alpha) in the sera of SLE-afflicted mice and in the sera and lymphoid tissues of 16/6Id-immunized BALB/c mice shortly after the immunization. We then found that administration of hCDR1 to 16/6Id-immunized mice specifically down-regulated SDF1alpha-induced T cell chemotaxis through fibronectin and collagen type I. This was accompanied by diminished SDF1-alpha-induced T cell adhesion and ERK phosphorylation. Treatment with hCDR1 up-regulated TGF-beta secretion, which, in turn, inhibited the murine T cell adhesion to and chemotaxis through fibronectin as well as their ERK phosphorylation. Thus, the secretion of TGF-beta after treatment of 16/6Id-immunized mice with hCDR1 plays an important role in the down-regulation of SDF-1alpha-mediated T cell activation and the interactions with extracellular matrix moieties observed in the present study.     

Proinflammatory interferon-gamma--inducing monokines (interleukin-12, interleukin-18, interleukin-15)--serum profile in patients with systemic lupus erythematosus

Concentrations of three interferon-gamma - inducing monokines, IL-12, IL-18 and IL-15, were investigated in the serum of 60 patients with systemic lupus erythematosus (SLE) and 20 healthy subjects. IL-12 and IL-18 were detectable in the serum of all patients with SLE and in all healthy controls. The level of IL-12 was significantly higher in SLE patients (median 264.9 pg/ml) than in the control group (median 163.6 pg/ml, p < 0.02). Similar differences were observed in the level of IL-18 (median values 602.2 pg/ml and 252.7 pg/ml, respectively, p < 0.001). Correlations between serum levels of IL-12 and IL-18 and SLE activity were not statistically significant (p > 0.05). We found a significant, positive correlation between IL-12 and IL-18 (rh? = 0.419, p < 0.001) in SLE patients. The level of IL-18 was higher in the SLE patients with antinuclear antibodies (ANA) (median 660.0 pg/ml) than in ANA-negative patients (median 326.5 pg/ml, p < 0.03), as well as in patients with immunoglobulin deposits at the dermal-epidermal junction (median 746.0 pg/ml and 444.0 pg/ml respectively, p < 0.04). The level of IL-12 was also higher in patients with skin immunoglobulin deposits (328.9 pg/ml) than those without this phenomenon (257.0 pg/ml, p < 0.05). The levels of both cytokines in the patients treated with immunosuppressive drugs and those patients not treated were similar. The serum levels of IL-15 were low and not significant both in SLE patients (median 2.9 pg/ml), and in healthy controls (median 1.6 pg/ml). In conclusion, serum levels of IL-12 and IL-18 are higher in SLE patients than in healthy controls which may indicate a role in the disease pathogenesis. However, they do not correlate with disease activity and seem to be unresponsive to immunosuppressive treatment.     

Strategies for production of monoclonal anti-idiotype antibodies against human B cell lymphomas

Murine monoclonal antibodies (MAB) against the idiotype (Id) of B lymphocyte malignancies are powerful reagents for the study of these diseases, and are potentially useful for treatment. Different strategies for the production of these anti-Id MAB have been compared. Initially, the Id Ig from nonsecreting B cell tumors was "rescued" by human X mouse or human X human hybridization. These somatic cell hybridizations resulted in the secretion of human Ig in 10 and 100% of the fusions, respectively. In a second step, anti-Id MAB were produced by using the "rescued" Id Ig as immunogen. A more streamlined approach is based on a one-step procedure in which the tumor cell suspension is used as immunogen. This method of immunization, coupled with a four-layer ELISA, results in the detection of anti-Id MAB in a frequency of approximately 1% of the total hybrids. By using a pool of 10 different anti-Id MAB, each reactive with the tumor of one patient, we searched for idiotypic relatedness among a panel of 50 additional tumors. No cross-reactions were found, indicating that our current strategy results in the identification of unique idiotypic determinants among human B cell tumors. Idiotypic Ig can be found in the serum of patients with B cell tumors. Among groups of patients, there is a wide spectrum of serum Id levels, ranging from less than 0.01 microgram/ml to greater than 500 micrograms/ml.     

Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.     

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants

BACKGROUND: Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. MATERIALS AND METHODS: Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. RESULTS: All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. CONCLUSIONS: Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a common anti-La/SSB idiotype. These antibodies can be detected using complementary peptides of La/SSB epitopes. The antiidiotypic antibodies mask the anti-La/SSB response. Hidden anti-La/SSB antibodies can be released and detected using complementary epitope analogs.