Robert A. Eisenberg

The lpr and gld genes in systemic autoimmunity: life and death in the Fas lane

The single gene lpr and gld models of spontaneous systemic autoimmunity have attracted much attention in recent years. Here, Philip Cohen and Robert Eisenberg describe the fascinating recent findings that the lpr and gld [corrected] phenotypes result from defects in the Fas gene and, perhaps, in the ligand for fas, respectively.

Circulating immune complexes in infective endocarditis.

To examine further the role of immune-complex deposition in infective endocarditis, we studied 29 patients with infective endocarditis for presence of complement-containing circulating immune complexes. Ninety-seven per cent (28 of 29) had serum levels of immune complexes greater than 12 mug per milliliter. Mean levels in these patients were significantly higher than in patients with sepsis without endocarditis or in normal controls (P less than 0.05). Circulating immune-complex levels were correlated with longer duration of illness (P less than 0.025), extravalvular manifestations of endocarditis (P less than 0.025) and hypocomplementemia (P less than 0.05). Patients with right-sided endocarditis had significantly higher circulating immune-complex levels than patients with left-sided involvement (P less than 0.025). In general, levels fell to zero with successful antimicrobial or surgical therapy. This drop was concurrent with disappearance of extravalvular signs, blood cultures becoming sterile, and rise in serum complement levels. These findings support the concept that immune complexes may be important in the pathogenesis of infective endocarditis.

A Phosphorus-Based Dendrimer Targets Inflammation and Osteoclastogenesis in Experimental Arthritis

A phosphorus-based dendrimer suppresses inflammation and reduces bone erosion in mouse models of rheumatoid arthritis. Dendrimer Doubles Up Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that is characterized by inflamed synovial tissue, cartilage degeneration, and bone erosion, leading to joint deformation and physical handicap. Biologic therapeutic approaches, such as monoclonal antibodies and soluble receptors, have been developed to relieve the symptoms of RA. Unfortunately, these drugs typically act on a single target: proinflammatory cytokines. There is an unmet clinical need for anti-RA drugs that target not only proinflammatory molecules and pathways, but also the osteoclastic differentiation of monocytes—a process that leads to bone resorption. In response, Hayder et al. synthesized a new dendrimer-based therapeutic that boasts a two-pronged attack on RA, with both anti-inflammatory and anti-osteoclastogenic activity. Dendrimers are highly branched polymers whose multivalency allows for interaction with several cellular and molecular targets. The authors created an azabisphosphonate (ABP)–capped dendrimer that has been shown to target human monocytes and direct them toward an anti-inflammatory response. Two animal models of autoimmune arthritis were used: the IL-1ra−/− mouse and the K/BxN serum transfer mouse. Dendrimer ABP was administered to IL-1ra−/− mice via weekly intravenous injections. At high doses, the dendrimer completely inhibited inflammation, as evidenced by a decrease in paw swelling, and prevented arthritis histopathology, with ankle joints showing near-normal synovial membranes and intact cartilage after 12 weeks. Dendrimer ABP also decreased the amount of proinflammatory cytokines and increased levels of anti-inflammatory cytokines interleukin-4 (IL-4) and IL-10, thereby suggesting a skewing toward a T helper 2 (TH2) response. The K/BxN mouse demonstrated similar results upon treatment with dendrimer ABP. Treatment with dendrimer ABP also prevented osteoclastogenesis, as shown in human synovial tissue ex vivo and human peripheral blood monocytes in vitro. The authors further outlined a potential dendrimer-mediated mechanism that involves inhibition of c-FMS—a signaling molecule that promotes monocyte differentiation into osteoclasts. This study by Hayder and colleagues has shown that dendrimer ABP, by doubling up against inflammation and bone erosion, might be more effective at treating RA than existing antibody- and small-molecule–based biologic drugs. Dendrimers are highly branched “tree-like” polymers that have demonstrated therapeutic potential in drug delivery, medical imaging, and tissue engineering in recent years. In addition, we have shown that an azabisphosphonate (ABP)–capped dendrimer selectively targets monocytes and directs them toward anti-inflammatory activation. We explored this property to assess the therapeutic potential of dendrimer ABP in the treatment of an inflammatory disease, rheumatoid arthritis. Intravenous injections of dendrimer ABP inhibited the development of inflammatory arthritis in two animal models: IL-1ra−/− mice and mice undergoing K/BxN serum transfer. Suppression of disease was characterized by normal synovial membranes, reduced levels of inflammatory cytokines, and the absence of cartilage destruction and bone erosion. Dendrimer ABP also exhibited anti-osteoclastic activity on mouse and human cells, mediated by c-FMS (cellular-feline McDonough strain sarcoma virus oncogene homolog) inhibition. These preclinical demonstrations suggest the potential use of dendrimer ABP as a nanotherapeutic for rheumatoid arthritis.

Stochastic control of anti-Sm autoantibodies in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr autoimmune mice consistently show an approximately 25% incidence of the systemic lupus erythematosus marker autoantibody anti-Sm. In the present report, we show that the failure to find anti-Sm antibodies in three-quarters of 5-mo-old MRL/lpr mice was not an artifact of an insensitive assay, but rather that the mice fell into two populations as regards their anti-Sm positivity. Based on an extensive analysis of the incidence of anti-Sm positivity in 5-mo-old mice according to their cage of residence, we found no evidence for genetic, environmental, or parental influences on the propensity of an individual animal to become anti-Sm positive. Also, the gender of the mouse, its Sm antigen level, or its length of survival were not related to anti-Sm antibody, nor was the anti-Sm antibody status of either parent. Some animals became anti-Sm positive after 5 mo of age, but this was less likely than becoming positive before 5 mo of age. Finally, a survey of 205 autoimmune C57BL/6-lpr/lpr mice confirmed the uniqueness of the MRL background for this autoantibody response. These results together indicate that the possibility of making anti-Sm antibodies is under genetic control, but that the expression of this capability in an individual animal is governed by stochastic events. We hypothesize further that such random processes may involve the expression of particular immunoglobulin variable-region genes combined with mechanisms of extensive somatic mutation or positive feedback amplification, which would transmute an initial monoclonal response into an eventual polyclonal one.

Subclass restriction and polyclonality of the systemic lupus erythematosus marker antibody anti-Sm

Anti-Sm antibodies are highly specific markers for the diagnosis of systemic lupus erythematosus (SLE). This specificity suggests that the immunoregulation of these autoantibodies would reflect fundamental immune abnormalities in this disorder. As a clue to this immunoregulation, we have investigated the isotype distribution of anti-Sm antibodies by enzyme-linked immunosorbent assays. We have found that the anti-Sm response is markedly restricted to the IgG1 heavy chain isotype. On the other hand, the light chain distribution reflects that in normal serum, while isoelectric focusing analysis fails to show an oligoclonal pattern. The related specificity, anti-ribonucleoprotein, is also restricted to IgG1, while the SLE-specific antibody anti-double-stranded DNA is mostly IgG1 with a lesser contribution by IgG3. These results suggest that antinuclear antibodies that are strongly associated with SLE are produced by a T cell-dependent response, probably driven by antigen. The immunoregulation of the response to several autoantigens may be quite similar.

Circulating anti-DNA-rheumatoid factor complexes in MRL1 mice

Abstract MRL 1 mice are a newly developed strain that spontaneously develops systemic lupus erythematosus, vasculitis, and arthritis characterized by high levels of anti-DNA antibodies and rheumatoid factors (RF). The majority of serum samples from 4- to 5-month-old MRL 1 mice contained IgG anti-DNA antibody complexes sedimenting in the intermediate position between 7 and 19 S markers as well as in the 7 S position. The intermediate anti-DNA antibody complexes were fully dissociated into 7 S IgG anti-DNA antibodies when serum samples were centrifuged in dissociating buffer (0.5 M acetate buffer, pH 4.0). Excess amounts of normal murine IgG inhibited reformation of the previously dissociated anti-DNA antibody complexes, and columns coated with the murine IgG selectively absorbed the intermediate anti-DNA antibody complexes. Furthermore, the intermediate anti-DNA antibody complexes were dissociable by the treatment with pepsin but not with DNase. Therefore, it was concluded that the anti-DNA antibodies sedimenting in the intermediate position were complexed with IgG RF without the involvement of DNase-sensitive materials. Although, in one group of sera, the concentration of anti-DNA-RF complexes in gradient fractions correlated well with those of all the IgG subclasses, in another group, only the IgG2a subclass was distributed in gradients proportionally to the anti-DNA-RF complexes, indicating the preferential reaction of IgG-RF with this particular subclass, Since most serum anti-DNA antibodies in MRL 1 mice belonged to the IgG2a subclass, IgG RF formed the complexes rather selectively with anti-DNA antibodies in the latter group of mice. Such complexes were rarely found in serum samples from other SLE-prone strains (NZB, NZB × W, and male BXSB). Consequently, these IgG anti-DNA-RF complexes, either by themselves and/or after the interaction of their anti-DNA antibodies with DNA antigens or their RF with diverse immune complexes, may be at least partially responsible for the spontaneously forming lesions in kidneys and other tissues of MRL 1 mice.

Experimental induction of systemic lupus erythematosus by recognition of foreign Ia.

A chronic GVH reaction induced in normal mice results in a syndrome that closely resembles SLE. In this study, we compared the autoimmune GVH syndrome induced in parent (C57BL/6Kh [B6] and B6.C-H-2bm12 [bm12]) and F1 [( B6 x bm12]F1) mice by transfer of parental spleen cells. A majority of the mice in all groups developed autoantibodies to chromatin and erythrocytes, and some of the mice also produced anti-dsDNA antibodies. The predominant isotype of the anti-chromatin autoantibodies was found to be IgG2a, although high levels of IgG2b antibodies were also present. Autoantibody production was in general more intense and more prevalent in parent----F1 hybrid combinations, compared to parent----parent infusions. No influence of host or graft gender was observed. These studies show that a chronic GVH reaction can be induced by both parent----parent and parent----hybrid combinations.

Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens

The Ku autoantigen is a DNA binding factor consisting of 70 and ∼80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factorsin vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of ∼1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610–705, 8–221, and 1–374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506–541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein. These mAbs constitute a panel of immunological reagents reactive with defined regions of the Ku autoantigen which should be useful for examining the assembly and function of Ku.

Quantitation and IgG subclass distribution of antichromatin autoantibodies in SLE mice.

Antibodies to a native chromatin preparation were found in most mice suffering from spontaneous SLE. All MRL/Mp-lpr/lpr (MRL/lpr) sera tested (more than 500) contained antibodies to chromatin and antichromatin levels increased with age. Approximately 50% of the IgG antichromatin antibody in the MRL/lpr sera was of the IgG2a subclass, 30% IgG2b, 10% IgG1, and 10% IgG3. Interestingly, the relative restriction of antichromatin autoantibodies to the IgG2a subclass was apparent in MRL/lpr mice as young as 1 month, well before the onset of lymphadenopathy. Antichromatin autoantibodies were also detectable in sera from MRL/Mp- +/+ (MRL/+), NZB, (NZB x NZW)F1 (B x W), and BXSB mice, but were not found in sera from normal mice. A similar subclass distribution skewed toward IgG2a was seen for MRL/+, B x W, and NZB mice. These results indicate that the spontaneous autoantibody directed against chromatin is a good marker for murine SLE, and is predominantly of the IgG2a subclass.

Class II major histocompatibility antigens and the etiology of systemic lupus erythematosus

It is hypothesized that the underlying immunoregulatory dysfunction in systemic lupus erythematosus (SLE) is altered recognition by T cells of self class II major histocompatibility antigens (Ia). The resultant cellular autoreactivity would directly cause certain of the immunopathological manifestations of SLE. The perception by T cells of self non-MHC antigens in the context of altered Ia on antigen presenting cells would also stimulate specific help for autoantibody production. Autoimmunity induced by the graft-versus-host reaction is an experimental model that illustrates this potential mechanism (A. G. Rolink, S. T. Pals, and E. Gleichmann, J. Exp. Med. 157, 755, 1983; R. A. Eisenberg, S. Y. Craven, and P. L. Cohen, Arth. Rheum. 26, S19, 1983).