Faculty Bibliography

Autoreactive antibodies that recognize neo-determinants on apoptotic cells in mice have been proposed to have protective, homeostatic and immunoregulatory properties, although our knowledge about the equivalent antibodies in humans has been much more limited. In the current study, human monoclonal antibodies with binding specificity for apoptotic cells were isolated from the bone marrow of healthy adults using phage display technology. These antibodies were shown to recognize phosphorylcholine (PC)-associated neo-determinants. Interestingly, three of the four identified apoptotic cell-specific antibody clones were encoded by VH3 region rearrangements with germline or nearly germline configuration without evidence of somatic hypermutation. Importantly, the different identified antibody clones had diverse heavy chain CDR3 and deduced binding surfaces as suggested by structure modeling. This may suggest a potentially great heterogeneity in human antibodies recognizing PC-related epitopes on apoptotic cells. To re-construct the postulated structural format of the parental anti-PC antibody, the dominant clone was also expressed as a recombinant human polymeric IgM, which revealed a substantially increased binding reactivity, with dose-dependent and antigen-inhibitable binding of apoptotic cells. Our findings may have implication for improved prognostic testing and therapeutic interventions in human inflammatory disease.

Objectives To study infection rates in patients with low immunoglobulin (Ig) serum concentrations following administration of rituximab (RTX) in RA clinical trials. Methods Pooled analysis of RA clinical trial data from patients who developed low IgM or IgG (defined as below lower limit of normal [LLN] for >4 mths [or 2 consecutive study visits]) after >1 RTX course. Igs were generally measured every 8-16 wks. Patients with low Ig were permitted to receive RTX re-treatment courses. Infection rates were assessed before and during/after low IgM/IgG. Low IgG/IgM at baseline screening (IgG <5.65 and IgM <0.55 mg/mL) were exclusion criteria for entry into the trials. Results Of 3194 patients who had received up to 17 RTX courses over 9.5 yrs, 22.4% (n=717) developed low IgM and 3.5% (n=112) developed low IgG for >4 mths. All had measurable Ig levels. No increases in overall infection rates were observed in patients during/after low IgM/IgG vs before documentation of low Ig (Table). For IgG, serious infection (SIE) rates were similar before and during/after low IgG, but both were significantly higher than in patients who never developed low IgG. At baseline these patients were on average older, had longer disease duration, lower mean CD19+ count, lower mean IgG levels (8.4 vs 13.2 mg/mL) and had received more non-biologic DMARDs vs those who did not develop low IgG. Baseline oral steroid use was similar across sub-groups. For IgM, SIE rates were not significantly higher during/after low IgM vs before low IgM, and were similar to rates in patients who never developed low IgM. In patients with low IgG or IgM, the SIE profile was consistent with RTX clinical experience as most infections affected the lower respiratory tract. Analysis of SIE onset in relation to timing of low Ig was limited due to discrete protocol-defined time points for Ig assessments. Other limitations included low patient numbers in some subgroups and lack of placebo comparator. (Table Presented) Conclusions Following RTX treatment, !

The adaptive immune system augments host defenses against diverse infectious threats, yet also carries intertwined risks for the development of autoimmune disease. The immune system incorporates homeostatic pathways for essential housekeeping functions that involve recognition of oxidation-modified endogenous molecules. Now, the properties of a physiological class of natural autoantibodies, which seem to modulate the severity or even prevent the onset of autoimmune disease, are beginning to be defined. Whereas disease-associated IgG autoantibodies to nuclear antigens and citrulline-modified self-proteins have been shown to activate innate pattern recognition receptors leading to increased cell death and tissue injury, a class of IgM autoantibodies to oxidation-associated neo-antigens can oppose these pathogenic effects. These naturally arising regulatory IgM autoantibodies enhance the capacity for the phagocytic clearance of host cells affected by programmed death pathways. These antibodies can also suppress key signalling pathways in the innate immune system involved in the control and resolution of inflammatory responses to Toll-like receptor agonists and disease-associated IgG autoantibodies.

The composition of the early immune repertoire is biased with prominent expression of spontaneously arising B cell clones that produce IgM with recurrent and often autoreactive binding specificities. Amongst these naturally arising antibodies (NAbs) are IgM antibodies that specifically recognized amaged and senescent cells, often via oxidation-associated neo-determinants. These NAbs are present from birth and can be further boosted by apoptotic cell challenge. Recent studies have shown that IgM NAb to apoptotic cells can enhance phagocytic clearance, as well as suppress proinflammatory responses induced via Toll-like receptors, and block pathogenic IgG-immune complex (IC)-mediated inflammatory responses. Specific antibody effector functions appear to be involved, as these anti-inflammatory properties are dependent on IgM-mediated recruitment of the early recognition factors of complement. Clinical surveys have suggested that anti-apoptotic cell (AC) IgM NAbs may modulate disease activity in some patients with autoimmune disease. In mechanistic studies, anti-AC NAbs were shown to act in dendritic cells by inhibition of the mitogen-activated protein kinase (MAPK) pathway, a primary signal transduction pathway that controls inflammatory responses. This immunomodulatory pathway has an absolute requirement for the induction of MAPK phosphatase-1. Taken together, recent studies have elucidated the novel properties of a class of protective NAbs, which may directly blunt inflammatory responses through a primitive pathway for regulation of the innate immune system.

Naturally arising IgM antibodies, which recognize neo-determinants on apoptotic cell (AC) membranes, are present from birth and can be further induced by AC challenge. Such naturally arising IgM antibodies can suppress proinflammatory responses to purified agonists for Toll-like receptors (TLRs), as well as block the induction of IgG immune complex-induced in vitro and in vivo pathogenic responses. To investigate the responsible mechanisms, we studied the regulatory effects of IgM anti-AC antibody on responses in bone marrow-derived dendritic cells mediated by a range of different TLRs and found that addition of IgM anti-AC inhibited the activation of the primary MAPKs: ERK1/2, JNK, and particularly p38. This was dependent on the recruitment of either C1q or mannose-binding lectin, which are both early complement factors that tag ACs for innate immune recognition. Strikingly, MAPK inhibition of responses to TLR agonists, and to lupus IgG autoantibody-chromatin immune complexes, was found to correlate with, and had an absolute requirement for, the induction and nuclear localization of MAPK phosphatase-1, a factor known to mediate glucocorticoid suppression of immune responses. Further experiments showed that natural IgM antibodies in serum exhibited the same inhibitory properties. These studies elucidate a novel homeostatic pathway by which natural antibodies, which are products of the adaptive immune system, can directly blunt inflammatory responses by recruitment and coordination of a primitive regulatory pathway of the innate immune system.

OBJECTIVE: Naturally arising IgM antibodies (NAb) to apoptotic cell (AC) determinants are present from birth and can be further induced by AC challenge. In systemic lupus erythematosus, lower anti-AC NAb levels have been associated with higher disease activity. We have recently shown that a prototypical AC-specific IgM NAb can suppress proinflammatory responses to purified agonists of Toll-like receptors and block the in vivo induction of IgG immune complex (IC)-induced arthritis. Nuclear antigens, which activate dendritic cells (DCs), form complexes with IgG autoantibody, and these have been implicated in the pathogenesis of autoimmune disease. In this study, we sought to investigate potential roles of such NAb for regulating IC-mediated activation of DCs, which is believed to be involved in disease initiation and perpetuation. METHODS: Bone marrow-derived myeloid DCs were stimulated with ICs composed of IgG autoantibody and chromatin or IgG autoantibody and RNA. Outcome was evaluated according to the production of inflammatory cytokines, as determined by enzyme-linked immunosorbent assay, and the expression of costimulatory molecules (markers of DC activation), as determined by flow cytometry. MAPK activation was evaluated by phospho-flow analysis and immunofluorescence microscopy. RESULTS: IgM anti-AC NAb dose-dependently suppressed the production of DNA IC- and RNA IC-induced interleukin-6 and DNA IC-induced tumor necrosis factor alpha, as well as the RNA IC-induced up-regulation of CD86 and CD40 on DCs. IgM NAb-mediated inhibition was associated with suppression of IC-mediated p38 MAPK activation and nuclear localization. CONCLUSION: We demonstrated a direct in vitro inhibitory effect of IgM NAb on inflammatory responses induced by IgG-nucleic acid ICs. These findings contribute to emerging evidence that regulatory NAb to AC determinants may oppose the influence of pathogenic lupus autoantibody ICs and thereby play roles in the maintenance of immune homeostasis.